CN109402064B - Hybridoma cell strain, monoclonal antibody produced by hybridoma cell strain and application of monoclonal antibody - Google Patents

Abstract

The present invention relates to the field of biotechnology, in particular to hybridoma cell lines and monoclonal antibodies produced therefrom and applications. The invention utilizes HUSSLCs to immunize BALB/C mice, establishes a functional monoclonal antibody library, and selects specific functional monoclonal antibodies that recognize and inhibit HUSSLCs from the library. Hybridoma cells produced by the fusion of HUSSLCs-sensitized B lymphocytes and myeloma SP2/0 cells can be screened and cloned to obtain a hybridoma cell line that stably secretes anti-HUSSLCs monoclonal antibody; the anti-HUSSLCs monoclonal antibody provided by the present invention can inhibit HUSSLCs Self-renewal, proliferation, migration, invasion, drug resistance.

Description

Hybridoma cell strain, monoclonal antibody produced by hybridoma cell strain and application of monoclonal antibody

Technical Field

The invention relates to the field of biotechnology, in particular to a hybridoma cell strain, a monoclonal antibody generated by the hybridoma cell strain and application of the hybridoma cell strain.

Background

Among all female reproductive malignancies, uterine sarcoma (uterine sarcoma) is a relatively rare one derived from connective tissue, uterine smooth muscle and the endometrial stroma. It can be classified into the following types according to the origin of tissue generation: of which 40% is uterine leiomyosarcoma (leiomyosarcoma); secondly, endometrial stromal sarcoma (endometeral sarcoma) accounts for 15%; the remainder are mixed epithelial and mesenchymal tumors, mixed mesenchymal tumors (iscellaneous mesenchyme tumors) and undifferentiated uterine sarcoma (undifferentiated uterine sarcoma), which are further classified into carcinosarcomas (carcinosarcomas) and adenosarcomas (adenosarcomas). Although the uterine sarcoma is rare, the incidence rate of the uterine sarcoma accounts for only 2-4% of the uterine malignant tumors, but the uterine sarcoma has high malignancy degree and poor prognosis, the 5-year survival rate is only 30% -50%, and the incidence rate of the uterine malignant tumor death causes is up to 25%. The important reasons for this are that most uterine sarcomas are not sensitive to radiotherapy and chemotherapy, have strong invasiveness and metastatic capacity, and are susceptible to recurrence. Therefore, finding out the main mechanism in the process of generating and developing the uterine sarcoma and finding out an effective method for treating the uterine sarcoma is a problem which needs to be solved urgently at present.

Disclosure of Invention

In view of the above, the invention provides a hybridoma cell line, a monoclonal antibody produced by the hybridoma cell line and application of the monoclonal antibody. According to the invention, HUSSLCs are used for immunizing a BALB/C mouse, a functional monoclonal antibody library is established, and a specific functional monoclonal antibody which can identify HUSSLCs and has an inhibiting effect on the HUSSLCs is screened out.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a hybridoma cell strain, the preservation number of which is CCTCC No. C2018182.

The invention also provides monoclonal antibodies selected from the group consisting of:

(I) the monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CCTCC No. C2018182;

(II) a monoclonal antibody capable of binding to an epitope of a monoclonal antibody produced by a hybridoma cell line having a preservation number of CCTCC No. C2018182;

(III) a monoclonal antibody that competes with a monoclonal antibody produced by a hybridoma cell line having a preservation number of CCTCC No. C2018182 in a competitive binding assay;

(IV) a monoclonal antibody having an antigen-binding fragment of the monoclonal antibody of any one of (I) to (III), wherein the fragment retains the ability to specifically bind to CD133 or a variant thereof.

On the basis, the invention also provides a preparation method of the monoclonal antibody, which takes human uterine sarcoma stem cell-like cells (HUSSLCs) as antigens to obtain B lymphocytes; fusing the B lymphocyte and SP2/0 cell to generate a hybridoma cell strain; screening to obtain positive hybridoma cells;

the human uterine sarcoma stem cell-like cells are suspension cell balls of a high-expression stem cell marker CD133 obtained from human uterine sarcoma SK-UT-1 cells.

Specifically, the preparation method of the human uterine sarcoma stem cell-like cell comprises the following steps:

(1) taking SK-UT-1 cells in logarithmic growth phase in SSM, making parent cells into suspension according to conventional method, centrifuging, discarding supernatant, adding serum-free culture solution, counting, inoculating into six-well ultra-low adhesion plate at 20000/ml density, placing in CO₂Culturing in an incubator. The day after, 1ml of serum-free medium was supplemented.

(2) Culturing in SFM for about 10 days, collecting suspension cell balls in a centrifuge tube, centrifuging, removing supernatant, adding 0.25% pancreatin, digesting at 37 deg.C for 5min, stopping digestion with culture solution containing 10% FBS, gently blowing cell suspension, centrifuging, washing, counting cells, adding SFM, keeping cell density at 20000/ml, and inoculating in a new six-well plate for culturing. The day after, 1ml of serum-free medium was supplemented.

The culture, passage, cryopreservation and recovery of the human uterine leiomyosarcoma cell line SK-UT-1 specifically comprise the following steps:

(1) SK-UT-1 cell line was routinely cultured in DMEM medium (SSM) containing 10% primary fetal bovine serum at 37 deg.C with 5% CO₂Good feelingCulturing in a humidity incubator, changing the culture solution after the adherence for 2-3 days, and carrying out passage once when the adherence fusion reaches 80% -90%.

(2) The culture medium of SK-UT-1 parental cells in logarithmic growth phase in SSM was discarded and the cells were washed 2 times with PBS. Adding pancreatin digestive juice (1ml/25 cm) into the culture flask²,2ml/75cm²) Digesting for 2-5min, observing under an inverted microscope, adding 2ml of culture solution to terminate digestion when the cell clearance is increased and the cell morphology begins to become round, centrifuging for 1000r multiplied by 5min, and discarding the supernatant. The cells were passaged 1:2, reseeded in a new flask and cultured.

(3) Preparing cell freezing stock solution, 10% DMSO, 70% DMEM, 20% FBS, preparing SK-UT-1 parent cell in logarithmic phase into single cell suspension, counting, centrifuging, removing supernatant, adding freezing stock solution to make cell concentration reach 5 × 10⁶And/ml, subpackaging in sterile freezing tubes, 1 ml/tube. Placing the frozen tubes in a 40 ℃ refrigerator for 30min, a-20 ℃ refrigerator for 2h, a-80 ℃ ultra-low temperature refrigerator overnight, and then placing the tubes in liquid nitrogen for storage.

(4) Taking out the frozen cells from the liquid nitrogen, immediately putting the frozen tube into a water bath at 37 ℃, shaking to melt the cells, taking out the frozen tube after the cells are completely melted, sterilizing the frozen tube with alcohol, sucking out the cell suspension in the frozen tube by using a suction pipe, transferring the cell suspension into a centrifuge tube added with 10 times of culture solution, centrifuging the cell suspension at a low speed of 1000rpm multiplied by 5min, removing the supernatant, and repeatedly washing the cell suspension with the culture solution once. Inoculating into culture flask at inoculation density of 5 × 10⁵/ml。

The stem cells are special cells in the body and have the remarkable biological characteristics of self-renewal and multidirectional differentiation. With the research on stem cells, people have increasingly advanced understanding on the occurrence and development of tumors, and theories that the tumors are derived from the stem cells, TSCs exist in the tumors and the like are provided. A small population of cells exists in tumors that are capable of self-renewal and can differentiate into tumor cells with various phenotypes, and is named TSCs because of these properties, which are similar to stem cell properties. The higher the content of TSCs in tumor cells, the higher the malignancy of the tumor, but at present, the research evidence can not prove whether all tumor tissues contain TSCs. In recent years, with the success of isolating more and more TSCs in tumor tissues, the identification of TSCs and the study of its biological properties have become a new focus, which points to a new direction for the study and treatment of malignant tumors. In 1997, Bonnet et al discovered that TSCs were present in AML when studying human Acute Myeloid Leukemia (AML) and could isolate these cells from AML. This is the first time that TSCs were discovered and isolated from human tumors in humans. This study found that a subpopulation of cells, representing 0.2% of total AML cells and having the phenotype CD34+ CD38-, possessed the ability to continue to form clones. After Non-obese diabetic/severer combined immunodeficiency (NOD/SCID) mice are inoculated with this fraction of cells, AML can be formed in the mice. Since the tissue structure characteristics of solid tumors are different from those of hematological tumors, the process of isolation and study of solid tumors is more difficult.

TSCs play an important role throughout the development of tumors. This importance is particularly evident in the context of tumor metastasis. Metastasis of tumors is a complex process involving multiple steps, with most malignant patients eventually dying from tumor metastasis. Tumor cells can migrate from their primary foci to other tissues and organs, and studies on their specific molecular mechanisms are still relatively superficial at present. The researchers have proposed the clone selection theory, which suggests that in the later stage of tumor development, part of tumor cells can obtain the ability of metastasis by mutation, and further make the tumor metastasize to other tissues and organs. It is also thought that the metastatic capacity of cells does not require late mutations, but rather exists initially, then translocates to other sites, guided by some signaling factors, and forms new tumor tissue when the external conditions are favorable. There is currently no strong evidence to support this hypothesis, but studies have long ago demonstrated that only one tumor cell is required to form a new tumor lesion. This ability to form and maintain tumors is only possessed by TSCs. Even if other tumor cells migrate to a new environment, they cannot differentiate indefinitely without self-renewal, and therefore cannot generate tumors in the new environment, and cannot maintain tumor growth and heterogeneity of tumor cells. In addition, compared with common tumor cells, TSCs are more genetically unstable and more able to adapt to and survive in new environments. Therefore, after the TSCs hypothesis was proposed, researchers have proposed a metastatic TSCs hypothesis, which suggests that some of the cells in TSCs have metastatic capacity, which is called metastatic TSCs, and the surrounding microenvironment of the metastatic TSCs is related to the size of their metastatic capacity.

After the study of TSCs resistance, many researchers found that tumor patients failed chemotherapy, mainly due to the presence of TSCs in tumor tissues. ABC transporters are expressed on cell membranes of most TSCs, and are involved in transportation and excretion of various substances including toxic substances, metabolites, drugs and the like. Neumanova Z et al found that ABCG2, a protein of human ABC, is involved in the formation of several barriers in the human body, including the blood testis, placenta and blood brain barrier. The study of Stacy AE et al found that ABCG2 is involved in cell metabolism and is called a "pump" because it can discharge metabolites, and ABCG2 can also protect the body from invasion of foreign substances. Because the ABCG2 has the special functions, the relation between the ABCG2 and the resistance of tumor cell chemotherapy drugs becomes a new research target of researchers in the aspect of anti-cancer treatment. The ABCG2 has the action mechanism that the energy required by discharging intracellular drugs is obtained by combining and hydrolyzing adenosine triphosphate, so that the concentration of the drugs in the cells is reduced, the toxic effect of the drugs on the cells is relieved, and the tumor cells generate drug resistance. The researchers use polymerase chain reaction to detect the expression of ABC protein in cells of AML patients before receiving chemotherapy, and find that the expression of leukemia stem cells ABCB1 and ABCG2 is closely related to the treatment effect of the patients, most of patients with high expression of ABCB1 and ABCG2 have no curative effect, and most of patients without expression are completely relieved. In AML cells, ABCB1 and ABCG2 were expressed more frequently, and resistance to daunorubicin was also stronger, so verapamil as an ABC transporter inhibitor could reduce such resistance. This could indicate that ABCG2 expression in AML stem cells correlates with resistance to AML chemotherapy. Therefore, a therapeutic approach targeting TSCs is expected to be the key point for reversing tumor resistance.

The existing anti-cancer therapy can not completely 'one-network' the tumor cells, and has an important reason which cannot be ignored. At present, the main anticancer therapy can only treat non-proliferative cells around the tumor, and cannot treat proliferative cells playing a key role in the tumor, namely TSCs. Therefore, the treatment of the tumor can only stay on the surface, but can not damage the root. Some drugs are effective mainly by reducing the tumor volume, and usually, only non-proliferating cells around the tumor are eliminated to effectively reduce the tumor volume. TSCs are similar to adult stem cells, possess most of their properties, and are better tolerated by chemotherapeutic drugs. After treatment with chemotherapeutic drugs, TSCs can stay in G0/G1 of the cell cycle, i.e., transition to a dormant state, thereby surviving attack by the chemotherapeutic drug and continuing to form new cancer cells after chemotherapy, resulting in recurrence of the cancer. It was found that after the traditional treatments, i.e. radiotherapy, chemotherapy and surgery, part of the patient's TSCs "by milling" still remained in the body, which had a viability hundreds of times higher than that of normal cancer cells and also had a higher metastatic capacity than normal cancer cells. The differentiation-promoting therapy is to make undifferentiated stem cells in tumor tissues differentiate to terminal cells and can make the tumor cells sensitive to cytotoxic drugs. Malumbres et al found that TSCs enter the metaphase of the cell cycle by inhibiting the activity of cyclin-promoting kinases which can cause chromosome and gene instability, thereby making tumor cells sensitive to chemotherapeutic drugs. Yanada et al found that acute promyelocytic leukemia can be cured by all-trans retinoic acid because all-trans retinoic acid can differentiate leukocytes, and that cytotoxic drugs in combination with all-trans retinoic acid can achieve better effects in treating patients in the late stage. Piccirillo et al found that bone morphogenic proteins, after binding to their own receptors, were able to differentiate adult glioma cells, significantly reducing the number of CD133+ cell populations, representing inhibition of TSCs growth.

In conclusion, TSCs play a critical role in the processes of metastasis, drug resistance and recurrence of malignant tumors, so that a treatment mode of targeting TSCs may open a promising and brand-new way for anticancer treatment.

The invention also provides application of the monoclonal antibody or the monoclonal antibody prepared by the preparation method in preparation of medicines and/or preparations for inhibiting self-renewal, unlimited proliferation, anchoring independent growth, tumorigenicity, migration, invasion and multidrug resistance of human uterine sarcoma stem cell-like cells (HUSSLCs).

The invention also provides the application of the monoclonal antibody or the monoclonal antibody prepared by the preparation method in preparing a medicament and/or a preparation for inhibiting the expression of the tumor stem cell marker CD133 protein.

The invention also provides the application of the monoclonal antibody or the monoclonal antibody prepared by the preparation method in preparing a medicament and/or a preparation for treating tumors.

The invention also provides the application of the monoclonal antibody or the monoclonal antibody prepared by the preparation method in preparing a reagent and/or a kit for detecting tumors.

In some embodiments of the invention, the tumor is uterine sarcoma.

The invention also provides a medicament which comprises the monoclonal antibody or the monoclonal antibody prepared by the preparation method and pharmaceutically acceptable auxiliary materials.

In some embodiments of the invention, the agent is capable of inhibiting self-renewal, unlimited proliferation, anchorage-independent growth, tumorigenicity, migration, invasion, or multidrug resistance of human uterine sarcoma stem cell-like cells (HUSSLCs).

In some embodiments of the invention, the medicament is capable of inhibiting the expression of the tumor stem cell marker CD133 protein.

The invention also provides a kit comprising the monoclonal antibody or the monoclonal antibody prepared by the preparation method.

In some embodiments of the invention, the kit is capable of detecting tumors.

The invention takes HUSSLCs as antigens to prepare a large amount of proliferated B lymphocytes; measuring the serum antibody titer by an ELISA method; fusing B lymphocyte with SP2/0 cell to generate hybridoma cell; screening positive hybridoma cells by an ELISA method; carrying out amplification culture on positive hybridoma cells by a limiting dilution method; detecting antibody subtype by an ELISA method; identifying the monoclonal antibody by an immunofluorescence method; the effects of monoclonal antibodies on HUSSLCs sphere formation, agar colony formation, wound healing, flow cytometry and Western blot assay were examined by the methods of sphere formation, agar colony formation, cell migration and expression of tumor stem cell marker proteins CD133, CD44, ABCG2, Bmi1, Nanog, Oct4 and ALDH 1.

The invention utilizes separated HUSSLCs to immunize a BALB/C mouse, establishes a functional monoclonal antibody library, screens out specific functional monoclonal antibodies which can identify HUSSLCs and have inhibition effect on the HUSSLCs, identifies the properties of the HUSSLCs and researches the functions of the HUSSLCs. The application of the HUSSLCs in the preparation of the HUSSLCs is expected to provide a candidate therapeutic agent with application value for the treatment of the targeted HUSSLCs, and lays a foundation for identifying a new molecular target of the HUSSLCs.

Benefits include, but are not limited to:

1. the titer of the antibody generated by the HUSSLCs immunized mouse meets the requirement of preparing the monoclonal antibody;

2. hybridoma cells generated by fusing HUSSLCs sensitized B lymphocytes and myeloma SP2/0 cells can be screened and cloned to obtain a hybridoma cell strain stably secreting anti-HUSSLCs monoclonal antibodies;

3. the anti-HUSSLCs monoclonal antibody can inhibit HUSSLCs from self-renewal, proliferation, migration, invasion and drug resistance;

4. after the second, third and impact immunity, the serum antibody titer is obviously increased (P is less than 0.05) compared with the first time;

5. SP2/0 cells were cultured for 3 days to 65% confluence, dead cells < 2%;

6. detecting hybridoma cells by an ELISA method to find positive holes;

7. after the cloning culture of the positive hole, 1 hybridoma cell strain which stably secretes the anti-HUSSLCs monoclonal antibody is obtained;

8. the monoclonal antibody subtype is IgG2 a;

9. the number of red fluorescent cells incubated by the supernatant of the hybridoma cell strain 1-2F is the largest;

10. 1-2F supernatant reduced HUSSLCs sphere formation, agar colony formation, cell mobility and expression of CD133, CD44, ABCG2, Bmi1, Nanog, Oct4, ALDH1 in a concentration-dependent manner.

Biological preservation Instructions

And (3) classification and naming: hybridoma cell line 1-2F; is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 08 and 14 months; the address of the preservation center is: wuhan university in Wuhan, China; the preservation number is CCTCC No. C2018182.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the detection of antibody titers in the sera (1:1000000) of HUSSLCs immunized mice by an indirect ELISA method;

FIG. 2 shows an image of SP-2/0 cell semi-adherent growth under an inverted phase contrast microscope (× 20);

FIG. 3 shows an image of adherent growth of a monolayer of fused hybridoma cells under an inverted phase contrast microscope (20);

FIG. 4 shows detection of positive hybridoma cell subtypes secreting specific antibodies by indirect ELISA;

FIG. 5 shows a cellular immunofluorescence image of monoclonal antibody detection HUSSLCs;

FIG. 6 shows that the 1-2F monoclonal antibody inhibits HUSSLCs sphere formation ability; note: a, contrast microscopy of a typical uterine sarcoma cytosphere image (x 100); b, determining the balling rate; control SP2/0 cell supernatant treated cells (Cont):^*P<0.05(Mean ± SD, n ═ 3); control 1-2F monoclonal cell supernatants (1:1000) treated cells:^#P<0.05(Mean±SD,n＝3)；

FIG. 7 shows the ability of the 1-2F monoclonal antibody to inhibit HUSSLCs agar colony formation; note: a, contrast microscopy of typical uterine sarcoma cell images (× 100); b, measuring agar colony forming rate; control SP2/0 cell supernatant treated cells (Cont):^*P<0.05(Mean ± SD, n ═ 3); control 1-2F monoclonal cell supernatants (1:1000)) And (3) treating the cells:^#P<0.05(Mean±SD,n＝3)；

FIG. 8 shows the ability of the 1-2F monoclonal antibody to inhibit cell migration of HUSSLCs; note: a, contrast microscopy of typical uterine sarcoma cell images (× 100); b, measuring the cell mobility; control SP2/0 cell supernatant treated cells (Cont):^*P<0.05(Mean ± SD, n ═ 3); control 1-2F monoclonal cell supernatants (1:1000) treated cells:^#P<0.05(Mean±SD,n＝3)；

FIG. 9 shows that the 1-2F monoclonal antibody reduces HUSSLCs CD133 positive cells; note: a, analyzing an image by a typical flow cytometer; b, CD133 positive cell percentage assay; control SP2/0 cell supernatant treated cells (Cont):^*P<0.05(Mean ± SD, n ═ 3); control 1-2F monoclonal cell supernatants (1:1000) treated cells:^#P<0.05(Mean±SD,n＝3)；

FIG. 10 shows that the 1-2F monoclonal antibody inhibits expression of HUSSLCs CD44 protein; note: a, typical Western blot banding pattern; b, Western blot analysis; control SP2/0 cell supernatant treated cells (Cont):^*P<0.05(Mean ± SD, n ═ 3); control 1-2F monoclonal cell supernatants (1:1000) treated cells:^#P<0.05(Mean±SD,n＝3)；

FIG. 11 shows that the 1-2F monoclonal antibody inhibits expression of HUSSLCs ABCG2 protein; note: a, typical Western blot banding pattern; b, Western blot analysis; control SP2/0 cell supernatant treated cells (Cont):^*P<0.05(Mean ± SD, n ═ 3); control 1-2F monoclonal cell supernatants (1:1000) treated cells:^#P<0.05(Mean±SD,n＝3)；

FIG. 12 shows that the 1-2F monoclonal antibody inhibits expression of HUSSLCs Bmi1 protein; note: a, typical Western blot banding pattern; b, Western blot analysis; control SP2/0 cell supernatant treated cells (Cont):^*P<0.05(Mean ± SD, n ═ 3); control 1-2F monoclonal cell supernatants (1:1000) treated cells:^#P<0.05(Mean±SD,n＝3)；

FIG. 13 shows that the 1-2F monoclonal antibody inhibits HUSSLCs Nanog protein expression; note: a, typical Western blot banding pattern; b, Western blot analysis; control SP2/0 cell supernatant treated cells (Cont):^*P<0.05(Mean±SD, n ═ 3); control 1-2F monoclonal cell supernatants (1:1000) treated cells:^#P<0.05(Mean±SD,n＝3)；

FIG. 14 shows that 1-2F monoclonal antibody inhibits HUSSLCs Oct4 protein expression; note: a, typical Western blot banding pattern; b, Western blot analysis; control SP2/0 cell supernatant treated cells (Cont):^*P<0.05(Mean ± SD, n ═ 3); control 1-2F monoclonal cell supernatants (1:1000) treated cells:^#P<0.05(Mean±SD,n＝3)；

FIG. 15 shows that the 1-2F monoclonal antibody inhibits expression of HUSSLCs ALDH1 protein; note: a, typical Western blot banding pattern; b, Western blot analysis; control SP2/0 cell supernatant treated cells (Cont):^*P<0.05(Mean ± SD, n ═ 3); control 1-2F monoclonal cell supernatants (1:1000) treated cells:^#P<0.05(Mean±SD,n＝3)。

Detailed Description

The invention discloses a hybridoma cell strain, a monoclonal antibody generated by the hybridoma cell strain and application of the monoclonal antibody. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Since Bonnet et al succeeded in isolating TSCs from human acute myelogenous leukemia in 1997, TSCs have been isolated from a variety of malignancies, such as breast cancer, pancreatic cancer, prostate cancer, and liver cancer, and a series of studies have shown that TSCs have close relationship with drug resistance, metastasis, and recurrence of malignancies, and thus, therapy targeting TSCs is expected to be a novel and effective therapy for malignancies. Existing targeted therapies for TSCs mainly include three types: molecular targeted therapy, differentiation-promoting therapy and antibody therapy.

Through research on various TSCs, the TSCs have multiple markers on the surface, wherein the CD133 is most widely distributed, such as liver cancer, brain cancer and colon cancer, and the tumorigenicity of CD133 positive cells is obviously enhanced compared with that of CD133 negative cells. The subject group successfully separates and cultures the suspension cell ball of the high expression stem cell marker CD133 from the uterine sarcoma cell line SK-UT-1 cells in earlier research, and experiments prove that the CD133 positive cell ball has obvious stem cell characteristics, and the proliferation, invasion, transfer and drug resistance capabilities are all stronger than those of parent uterine sarcoma cells. Thus, CD133 is promising as a therapeutic target for uterine sarcoma stem cell-like cells.

The animals can produce antibodies after being stimulated by antigens, the specific antibodies are directed to a specific determinant on the surface of the antigens, and the antibodies produced by the immunized animals are a mixture of a plurality of antibodies due to the diversity of the determinants on the surface of the antigens, so that the separation of different antibodies is extremely difficult, and the amount of the obtained antibodies is small. The monoclonal antibody technology has solved the problem well. In 1975, Milstein and Kohler invented monoclonal antibody technology, which is based on the following principles: antibodies are produced by only one type of cell. B lymphocytes, although producing antibodies, are unable to divide indefinitely in vitro; while tumor cells can be passaged indefinitely in vitro, they cannot produce antibodies. Therefore, the hybridoma obtained by fusing these two cells has both the characteristics of two cells and can produce an antibody while dividing indefinitely. The preparation of the monoclonal antibody comprises the following steps: first, immunizing a mouse with an antigen to obtain a plurality of proliferated B lymphocytes capable of secreting antibodies; secondly, fusing tumor cells with an unlimited passage function with B lymphocytes to generate hybridoma cells; thirdly, obtaining hybridoma cells only producing specific antibodies through screening; fourthly, there are two methods for the production of monoclonal antibodies: one is to culture hybridoma cells in vitro in large scale and then obtain monoclonal antibody from hybridoma cell culture solution; the other method is to inject hybridoma cells into abdominal cavity of the mouse and then obtain the monoclonal antibody from the abdominal cavity of the mouse. As an important tumor treatment mode, researchers have prepared corresponding monoclonal antibodies by separating and purifying tumor stem cells so as to provide effective candidate antibody medicaments for targeted therapy of the tumor stem cells. Researchers obtain anti-liver cancer TSCs monoclonal antibodies co-localized with the surface marker CD133 of the liver cancer TSCs, and in vitro studies prove that the monoclonal antibodies can obviously inhibit the proliferation and the balling growth of the TSCs.

The invention utilizes the separated CD133 positive uterine sarcoma stem cell-like cells to prepare the monoclonal antibody which targets the uterine sarcoma stem cell-like cells and has obvious inhibition effect on stem cell characteristics of proliferation, invasion, transfer and the like of the uterine sarcoma stem cell-like cells, and is expected to become a new and valuable treatment mode of the uterine sarcoma.

Preparation and characterization of monoclonal antibodies

BALB/c mice have good immune conditions, and the immunity is gradually reduced along with the increase of the age of the animals, so that female mice are more convenient to operate compared with male mice, and female BALB/c mice with the age of 6-8 weeks are preferred as immune animals. Experiment 2.4.1.1 sera from 6 week old female BALB/c mice immunized with HUSSLCs were tested for antibody titer by indirect ELISA. The negative control group is the serum of the unimmunized mouse, and whether the result is positive is judged by taking 2.1 times of the antibody titer of the negative control group as a critical value. The experimental results show that: antibody levels in mice, with lower potency after the first immunization; after the second, third and impact immunization, the antibody titer is obviously increased, which shows that HUSSLCs can cause stronger immune reaction in mice and have good immunogenicity. The indexes for evaluating the good immune effect when preparing the monoclonal antibody are as follows: the serum titer reaches more than ten thousand. Reading by a microplate reader, and selecting an antiserum dilution multiple with an absorbance value of 0.7-1.2 to obtain the antibody titer. Our experimental data show that after the second immunization, the absorbance values of the mouse serum antibodies in million-fold dilution meet the range, which is far more than the preparation requirement of the monoclonal antibodies.

Tumor cell lines such as P3, SP2/0, NSO/1 and the like are homologous with BALB/c mice, and the growth and fusion of the SP2/0 cell line are the most dominant, so the tumor cell line which is most widely applied at present is myeloma SP 2/0. Myeloma cells must be maintained in a very good state prior to fusion to ensure a high fusion rate, and growth to a density of about 60-80% is generally considered to be in a log-tonic phase. The myeloma cells cultured by the invention can reach 65% density after being cultured for 3 days, and the counting result of trypan blue staining cells proves that: the percentage of dead cells is less than 2%; thus, SP2/0 cells have high viability.

Cell fusion methods are mainly classified into three types: the induction efficiency of the biological method is low, the induction is unstable and the repeatability is poor; the physical method has high cost and complex operation although the cost is high; we chose a simple, cost-effective chemical approach, using polyethylene glycol as the cell fusion agent. The present invention uses PEG1500 as a cosolvent for cell fusion using chemical methods. The preparation process of the monoclonal antibody comprises two times of screening, wherein hybridoma cells are screened for the first time, and hybridoma cells capable of generating specific antibodies are screened for the second time. The first screen selective culture of hybridoma cells after cell fusion is critical. HAT selective medium is prepared by adding hypoxanthine (H), methotrexate (A) and thymidine (T) to common culture medium of animal cells. It is based on two pathways for DNA synthesis in cells: one pathway is the "D pathway" (biosynthetic pathway), which is the synthesis of nucleotides from amino acids and other small molecule compounds, providing a starting material for the synthesis of DNA molecules. Folic acid is involved in this process as an important coenzyme, while methotrexate is a folate antagonist in HAT cultures and blocks the "D-pathway". The other path is an 'S path' (emergency path), which utilizes hypoxanthine-guanine nucleoside phosphate transferase and thymidine kinase to catalyze hypoxanthine and thymidine to generate corresponding nucleotides, and one of the two enzymes is not necessary. Therefore, in HAT culture medium, the "D pathway" of unfused B lymphocytes and self-fused cells of two B lymphocytes is blocked by methotrexate, and the "S pathway" is normal, but the cells lack the ability to proliferate in vitro culture medium, and die in general in about 7 to 10 days. Myeloma cells and self-fused cells do not have the "S pathway" itself because they are deficient in hypoxanthine-guanine nucleoside phosphate transferase, and the "D pathway" is blocked by methotrexate, and therefore they cannot proliferate in HAT culture medium and die quickly. Only the hybridoma cells formed by fusing myeloma cells and B lymphocytes with each other have both the "S pathway" of B lymphocytes and the characteristic that myeloma cells proliferate in vitro for a long period of time, and therefore can selectively survive and proliferate in HAT culture medium. And (3) screening for 8 days by using a liquid culture medium culture method, clustering and growing the fused hybridoma cells to form small cell colonies, uniformly mixing and brightening the cells, and having high proliferation rate.

The invention discovers that 6 positive holes exist by screening positive hybridoma cells detected by an indirect ELISA method. Cloning the 6 positive wells by a limiting dilution method, and obtaining 6 hybridoma cell strains which stably secrete anti-HUSSLCs monoclonal antibodies through 4 times of cloning. The supernatants of cell lines 1-2F were found to exhibit the most red fluorescence by immunofluorescence detection. The monoclonal antibody is bound with the HUSSLCs through a specific target point on the surface, and because the monoclonal antibodies screened by the monoclonal antibodies are IgG antibodies, the anti-IgG secondary antibody with the fluorescent label can be bound with the monoclonal antibodies. By washing away the mabs that failed to bind to HUSSLCs, the mabs that eventually showed red fluorescence were those that bound to HUSSLCs. Therefore, the monoclonal antibody secreted by the 1-2F cell strain provided by the invention is most probably a functional monoclonal antibody capable of specifically binding with HUSSLCs, so that in the following functional experiments, the 1-2F monoclonal cell strain is selected.

In vitro functional study of anti-HUSSLCs monoclonal antibody

HUSSLCs sphere formation experiment

Unlike normal cells, tumor cells do not undergo contact inhibition, particularly TSCs, and therefore are more likely to spherodize. The balling capacity is an important method for TSCs in vitro functional experiments, and can judge the self-renewal capacity of single cells in a proper conditioned medium, and is generally expressed by the balling formation rate of the cells. Measurement of the rate of formation of HUSSLCs spheres: the 1-2F monoclonal cell supernatant at different dilution ratios was found to reduce the HUSSLCs sphere formation rate in a concentration-dependent manner, indicating that the 1-2F monoclonal antibody has the effect of inhibiting HUSSLCs self-renewal.

HUSSLCs agar colony formation assay

Tumor cells can be immortalized in soft agar medium to form cell colonies, while mature differentiated cells lose their ability to form colonies in soft agar. Agar colony formation is commonly used to detect the anchorage-independent growth and anoikis apoptosis-resistant ability of tumor stem cells and is positively correlated with the in vivo tumorigenic potential of tumor cells. The agar colony formation results of this experiment show that different dilution ratios of 1-2F monoclonal cell supernatants reduce the HUSSLCs agar colony formation rate in a concentration-dependent manner. The 1-2F monoclonal antibody is shown to have the effect of inhibiting HUSSLCs anchorage-independent growth and tumorigenicity.

HUSSLCs cell migration assay

Research shows that the tumor stem cells present an epithelial-mesenchymal transition (EMT) phenotype and have stronger in vitro cell migration, invasion and in vivo transfer capacities. The result of the wound healing method for measuring the cell mobility shows that the cell mobility of HUSSLCs is reduced by the supernatant of the 1-2F monoclonal cells with different dilution ratios in a concentration-dependent manner. The effect of the 1-2F monoclonal antibody on inhibiting the migration and movement of HUSSLCs is shown.

HUSSLCs CD133 expression assay

CD133 is a transmembrane glycoprotein, is an important surface marker of stem cells of various tissues including liver cancer and uterine sarcoma and tumor stem cells thereof, and is associated with poor prognosis of patients with various types of tumors. HUSSLCs used in the experiment are human uterine sarcoma stem cell-like cells which are separated in the previous experiment and highly express CD 133. Flow cytometry results of PE-labeled CD133 antibody showed that different dilution ratios of 1-2F monoclonal cell supernatant reduced the percentage of HUSSLCs CD133 positive cells in a concentration-dependent manner. The 1-2F monoclonal cell culture medium supernatant is proved to have the effect of inhibiting the expression of the HUSSLCs tumor stem cell marker CD133 protein or effectively reducing the number of tumor stem cells.

Western blot experiments for expression of HUSSLCs surface marker proteins CD44, ABCG2, Bmi1, Nanog, Oct4 and ALDH1

The research finds that the over-expression of the CD44 can promote and maintain the self-renewal, migration and invasion of the tumor stem cells and is a marker protein of various tumor stem cells; ABCG2 overexpression contributes to and maintains tumor stem cell multidrug resistance; the abnormal expression of Bmi1 promotes and maintains the self-renewal, unlimited proliferation and multidrug resistance properties of tumor stem cells; the abnormal expression of Nanog is related to the self-renewal and unlimited proliferation functions of tumor stem cells; aberrant expression of Oct4 correlates with tumor stem cell self-renewal, unrestricted proliferation function, and EMT phenotype; abnormal expression of ALDH1 enhances the cytotoxic effects of stem cell and tumor stem cell resistance to drugs and other compounds, and contributes to and maintains tumor stem cell multidrug resistance and cellular anoikis resistance. The results of western blot analysis of this experiment showed that different dilution ratios of 1-2F monoclonal cell supernatant reduced the expression level of HUSSLCs CD44, ABCG2, Bmi1, Nanog, Oct4, ALDH1 protein in a concentration-dependent manner (P < 0.05). These results show that the 1-2F monoclonal antibody has the function of inhibiting the expression of a plurality of tumor stem cell markers above HUSSLCs, and the monoclonal antibody can effectively inhibit the self-renewal, unlimited proliferation, migration, invasion, multidrug resistance and other tumor stem cell characteristics of HUSSLCs.

At present, no research report of a functional monoclonal antibody aiming at the uterine sarcoma stem cells is seen at home and abroad. The subject group is currently performing the next in vivo functional test, using cell tumorigenic nude mice containing different tumor markers, and then using positive monoclonal antibodies to perform antibody therapy experiments on the tumorigenic nude mice. Further study on whether the functional monoclonal antibodies can effectively inhibit the growth and the metastasis of the uterine sarcoma transplantable tumor and prolong the life cycle of tumor-bearing mice. The application of the HUSSLCs in the preparation of the HUSSLCs is expected to provide a candidate therapeutic agent with application value for the treatment of the targeted HUSSLCs, and lays a foundation for identifying a new molecular target of the HUSSLCs.

The hybridoma cell strain, the monoclonal antibody produced by the hybridoma cell strain, and raw materials and reagents used in application of the hybridoma cell strain can be purchased from the market.

Cell line and experimental animal

The 2 nd generation CD133+ spherical cells (named HUSSLCs) of the SK-UT-1 cell line are from earlier experiments and stored in the cell center of Hunan ya Hospital.

Myeloma SP2/0 cell line cell, offered as a gift by professor Liu Shi of university of Hunan province, was cryopreserved in the laboratories of the pharmaceutical center of university of Hunan province.

5-week-old female Balb/c mice: purchased from laboratory animal technology, ltd, viton, beijing, and all animal experiments were approved by the ethical committee on laboratory animals of the medical college of university of faculty, hunan.

Main instruments and articles

Name (R)	Source company
		Carbon dioxide cell incubator	American SHEL-LAB Co
Medical purification workbench	Suzhou clarification plant Co Ltd
		Inverted microscope	Olympus, Japan Ltd
Vortex oscillator	Seientifischidvseries of USA
		High-speed centrifugal machine	SHANGHAI ANTING SCIENTIFIC INSTRUMENT FACTORY
Electronic constant temperature water bath	China Beijing Zhongxing Weiwei Instrument Co Ltd
		Micro-pipette	Eppendorf Co, Germany
Cell blowing and beating pipe	Vial Ltd
		Electric heating constant temperature incubator	Shanghai Jinghong experiment equipment Co Ltd
Cell culture dish	Corning Inc. of USA
		Cell culture plate	Corning Inc. of USA
Cell culture bottle	Corning Inc. of USA
		Stainless steel cell sieve	Shanghai Biobiol Ltd
Decoloration shaking table	Jiangsu gold Tan Co Ltd
		Scanner Ranon GIS-2008	Shanghai Tian energy science & technology Limited
Voltage-stabilizing current-stabilizing electrophoresis apparatus	Beijing Liuyi Instrument Factory
		Membrane rotating instrument	Bio-Rad Inc. of USA

Main experimental reagent

English abbreviation

Statistical method

The experimental data are expressed as Mean ± standard deviation (Mean ± SD), and variance was analyzed using SPSS (statistical products and service solutions software) 18.0 statistics, ANOVA. The comparisons between the homogeneous variances are performed using LSD, and the heterogeneous sets of means are tested using Tukey's test. P <0.05 differences were statistically significant.

The invention is further illustrated by the following examples:

example 1 establishment of a Large-Capacity anti-HUSSLCs monoclonal antibody library, screening and identification of monoclonal antibodies capable of specifically recognizing HUSSLCs

Indirect ELISA method for detecting serum titer of HUSSLCs immune Balb/c mice

Mouse immunization test

2 female BALB/c mice of 6 weeks old were injected with HUSSLCs 1X 10 per abdominal cavity⁷0.5ml of physiological saline; after 2 weeks of the primary immunization, the secondary immunization was performed according to the primary immunization method; after 2 weeks of the second immunization, the third immunization was performed according to the primary immunization method; after 2 weeks of the third immunization, each mouse spleen was injected with HUSSLCs 5X 10⁵0.1ml of physiological saline for impact immunization. After 3 days, spleen cell suspensions were prepared.

Indirect ELISA method for detecting antibody titer of serum

(1) Serum titers were measured 2 weeks after each immunization by taking blood from the orbital wells of the mice. The mouse head was held down, the inner canthus of the mouse was inserted with a capillary, 15. mu.l of blood was collected, transferred to a small centrifuge tube, 3000 rpm, and centrifuged for 10 minutes. An appropriate amount of plasma was aspirated by a sample injector, and the serum was diluted with PBS at a concentration gradient of 1:10, 1:100, 1:1000, 1:10000, 1:100000, 1: 1000000. Then, enzyme-linked immunosorbent assay was performed.

(2) Adding 50 mul of 10 mug/ml L-polylysine into each well of a 96-well cell culture plate, standing at room temperature for 30 minutes, and washing with PBS for 2 times; add 50. mu.l HUSSLCs cell suspension (2.5X 10) per well⁶Cells/ml), overnight at 4 ℃, washed 1 time the next day with PBS; add 50. mu.l of 0.5% glutaraldehyde into each well, fix for 15 minutes at 4 ℃ and wash 2 times with PBS; adding 400 μ l of 0.1mol/l glycine solution into each well, standing at 4 deg.C for 30min, and washing with PBS for 3 times; adding incomplete DMEM medium into each well, and storing at-20 deg.C for use; during detection, the cell culture plate is taken out from a refrigerator at the temperature of-20 ℃, and after liquid in the hole is melted, the cell culture plate is washed for 2 times by PBS; adding 100 μ l of immune mouse plasma to be tested into each well, repeating each gradient with three wells, standing at 37 deg.C for 2 hr, and washing with PBS 4 times; adding 100 μ l enzyme-labeled goat anti-mouse IgG antibody into each well, acting at 37 ℃ for 2 hours, and washing with PBS for 6 times; adding 200 mul of newly prepared substrate TMB into each hole, and acting for 10 minutes at 37 ℃; adding 50 mul of stop solution into each hole to stop the reaction; the absorbance values were measured at a wavelength of 450nm using an enzyme-labeled spectrophotometer.

In vitro culture of myeloma SP2/0 cell line cells

According to the literature^[29]The method of (4), SP2/0 cell line is maintained in 1640 cell culture medium containing 100. mu.g/ml of streptomycin and 100IU/ml of penicillin G, and 10% FBS. The cells were placed in an incubator at 37 ℃ and 5% carbon dioxide to allow semi-adherent growth of SP2/0 cells, and expanded routinely at 1 passage every 2 days (1: 4).

Cell fusion

(1) 1 unimmunized BALB/c mouse was sacrificed and the mice were sterilized after 5 to 10 minutes of immersion in 75% alcohol; in the clean bench, mice were supine on the dissection dish. Cutting the abdominal skin of the mouse with a pair of sterilizing scissors, pulling the skin along the incision, and sterilizing with alcohol again; 5ml of serum-free 1640 culture solution was injected into the abdominal cavity with a syringe, and after gently kneading the abdominal cavity for 1 to 2 minutes, the liquid in the abdominal cavity of the mouse was aspirated with a syringe. The intraperitoneal fluid was transferred into 20% FBS-HAT-1640 medium (400 ml).

(2) Taking 1 mouse which is subjected to HUSSLCs impact immunization, taking off eyeballs of the mouse, bleeding, killing the mouse, soaking the mouse in 75% alcohol for 5-10 minutes, and then disinfecting the mouse; in a superclean workbench, a mouse lies on a dissecting plate in a supine position, the spleen of the mouse is cut off, surrounding fat and connective tissue are removed, and the spleen is washed for a plurality of times by serum-free culture solution; placing the spleen into a cell sieve, adding 3ml of serum-free 1640 culture solution into a culture dish, transferring the cell sieve into the culture dish, grinding the spleen, cleaning spleen tissues by using the serum-free culture solution, and collecting cell suspension. 1000g of the spleen cell suspension is centrifuged for 2 minutes, the supernatant is removed, the spleen cells are resuspended in a serum-free 1640 culture solution, and the spleen cells are repeatedly washed until no bulk particles are formed.

(3) Well-growing SP2/0 cells were selected, SP2/0 cells were washed 3 times with serum-free 1640 medium, and SP2/0 cells and treated spleen cells were mixed together at the last wash. Centrifuging 1000g of the cell mixed solution for 2 minutes, and removing a supernatant; adding the precipitate into 1ml of 37 deg.C PEG1500, blowing and beating the cells to mix them uniformly, adding 30ml of serum-free culture solution after 1 minute, and stopping cell fusion; then, centrifuging 1000g of the fused cell mixed solution for 2 minutes, and removing the supernatant; the pellet was resuspended in the prepared 20% FBS-HAT-1640 medium containing feeder cells. Inoculating the fused cells into a 96-well culture plate according to 200 mul/well, placing the culture plate in an incubator at 37 ℃ and 5% carbon dioxide, and observing the growth state of the cells every day; the culture medium was changed 1 time for 2 days, and 2/3 of old culture medium was removed each time and an equal amount of fresh culture medium was added. After 8 days of culture, the cells were cultured in HT medium, and when the cell colonies grew to 1/3-1/2 in the culture well, the next experimental procedure was carried out.

Screening of Positive hybridoma cells by ELISA method

(1) And (3) taking 50 mu l/hole of hybridoma cell culture supernatant, adding the hybridoma cell culture supernatant into an enzyme label plate, wherein a negative control is to select 2 holes without cell growth in each plate, and a positive control is to select 2 holes without cell growth to add positive serum. The microplate was incubated at 37 ℃ for half an hour.

(2) 100ul enzyme-labeled secondary antibody was added to each well and incubated at 37 ℃ for half an hour.

(3) 200ml of substrate was added to each well and developed for half an hour at 37 ℃.

(4) Detecting absorbance value (OD value) at 450nm wavelength with enzyme-labeled spectrophotometer, comparing OD value of negative control hole, and using P/N greater than 2.1 as critical point.

Cloning and culturing positive hybridoma cell by limiting dilution method

(1) After cell fusion and selective culture are successful, taking a cell hole with ELISA detection as positive, and preparing a cell suspension;

(2) the cell suspension was prepared as follows 1: 2. 1: 4. 1: 8. 1: 16. 1: 32, and inoculating into 96-well plates, repeating for 8 wells for each cell concentration;

(3) observing the growth state of the cells, and changing the liquid every two days;

(4) when the cell fusion degree reaches 70%, taking the supernatant and carrying out positive detection by adopting ELISA;

(5) selecting positive single-set falling holes, and repeating the steps 1-4 until the positive rate reaches 100%;

(6) culturing positive clone cells with common 24-well plate, and transferring to 25cm²And culturing and amplifying in a culture flask.

Detection of monoclonal antibody subtype by indirect ELISA method

(1) The 96-well cell culture plate was added with 50. mu.l of 10. mu.g/mL L-polylysine per well, left at room temperature for 30 minutes, and then washed 2 times with PBS.

(2) Add 50. mu.l HUSSLCs cell suspension (2.5X 10) per well⁶cells/mL). 4 ℃ overnight, the next day with PBS washing 1 time.

(3) Mu.l of 0.5% glutaraldehyde was added to each well, fixed at 4 ℃ for 15 minutes, and washed 2 times with PBS.

(4) Mu.l of 0.1mol/l glycine solution was added to each well, and the mixture was left at 4 ℃ for 30 minutes and washed 3 times with PBS.

(5) Adding incomplete DMEM medium each time, and storing at-20 deg.C for use.

(6) During detection, the cell culture plate is taken out from a refrigerator at the temperature of-20 ℃, and after liquid in the hole is melted, the cell culture plate is washed for 2 times by PBS.

(7) Mu.l of hybridoma cell supernatant to be tested was added to each well, left at room temperature for 2 hours, washed 4 times with PBS, and incubated at 37 ℃ for 1 hour.

(8) Mu.l of enzyme-labeled secondary antibody (goat anti-mouse IgM, goat anti-mouse IgG1, goat anti-mouse IgG2a, goat anti-mouse IgG2b, and goat anti-mouse IgG3) was added to each well, and the mixture was allowed to react at room temperature for 2 hours, washed 6 times with PBS, and incubated at 37 ℃ for 30 min.

(9) 200. mu.l of the freshly prepared substrate TMB was added to each well and allowed to act for 30 minutes at 37 ℃.

(10) The absorbance values were measured at a wavelength of 450nm using an enzyme-labeled spectrophotometer.

Identification of monoclonal antibodies by indirect immunofluorescence experiments

(1) Taking HUSSLCs, and inoculating the HUSSLCs into a common 6-hole plate;

(2) after the cell fusion degree reaches 70%, rinsing with PBS for 2 times, and fixing with 4% paraformaldehyde at room temperature for 10 min;

(3) rinsing with PBS for 3 times, 3min each time, and incubating hybridoma cell culture supernatant at 37 ℃ for 1 h;

(4) rinsing with PBS for 3 times (3 min each time), and incubating at 37 deg.C for 1h with goat anti-mouse-cy 7-fluorescent secondary antibody (diluted at 1: 500);

(5) rinsing with PBS for 3 times (3 min each time), incubating with fluorescent dye DAPI (1:100, diluted with PBS) at room temperature in dark for 10min, rinsing with PBS for 3 times (3 min each time); and (4) observing under a microscope.

As a result:

detection of HUSSLCs immunogenicity by indirect ELISA method

Sera from HUSSLCs immunized mice were tested for antibody titer by indirect ELISA. The negative control group is the serum of the unimmunized mouse, and whether the result is positive is judged by taking 2.1 times of the antibody titer of the negative control group as a critical value. The immune serum experimental results (table 1, fig. 1) show: the antibody level in the body of the mouse is low after the first immunization, and has no obvious difference with a negative control; after the second, third and impact immunizations, the antibody titer was increased significantly, with P <0.05, and the difference was statistically significant.

TABLE 1 Indirect ELISA method for determining the antibody titre of the serum (1:1000000) of HUSSLCs immunized mice

Note:^*: the first mouse serum antibody titer was compared with the pre-immunization, and the difference was statistically significant (P)<0.05)；^#: the serum antibody titer of the second mouse was compared with that of the mice before immunization, and the difference was statistically significant (P)<0.05)

Semi-adherent growth of SP2/0 myeloma cells

Myeloma SP2/0 cell line cells grew semi-adherent in complete 1640 medium with 10% FBS (FIG. 2), were round, morphologically intact, and aligned, and reached a density of 65% after 3 days of culture.

Cell fusion and positive hybridoma cell screening

After 8 days of screening and culturing by a liquid medium culture method, the fused hybridoma cells grow into clusters to form small cell colonies, the cells are mixed and are bright, and the cells proliferate quickly (as shown in figure 3). Positive hybridoma cells were detected by indirect ELISA, and 6 positive wells were found, and the numbers of the culture wells were designated 4-2, 9-15, 10-2, 10-4, 10-11, and 10-12, respectively.

Cloning and culturing positive hybridoma cell by limiting dilution method

After cloning the positive wells for 4 times by a limiting dilution method, an indirect ELISA method finds that 1 hybridoma cell strain can stably secrete the anti-HUSSLCs monoclonal antibody, and the monoclonal antibody is named as 1-2F. And (5) freezing and storing by using conventional liquid nitrogen.

Determination of monoclonal antibody subtype by indirect ELISA method

The indirect ELISA results show that: the subtype of antibody secreted by cell line 1-2F was IgG2a (FIG. 4).

Identification of monoclonal antibodies by indirect immunofluorescence experiments

The indirect cell immunofluorescence detection result shows that HUSSLCs incubated by the supernatant of the 1-2F monoclonal cells show red fluorescence in different degrees. And the number of red fluorescence positive cells incubated with the supernatant of 1-2F monoclonal cells was the greatest (FIG. 5).

Example 2 Balling experiment to examine the Effect of monoclonal antibodies on HUSSLCs Balling

(1) The supernatant of SP2/0 cells and the supernatant of positive hybridoma cells (1:1000, 1:500, 1: 100) at different dilution ratios were exposed to HUSSLCs for 48 hours.

(2) Balling culture: according to the literature^{[27，28，29]}According to the method, a stem cell culture medium is prepared, and EGF and bFGF 20ng/ml, insulin 5 mu g/ml, a B27 supplement and 0.4% BSA are added into a serum-free DMEM/F12 culture medium. HUSSLCs cells were suspended, adjusted to a density of 10000 cells/ml and the cell suspension was seeded onto 6-well ultra low-stick plates, 2.0 ml/well. Fresh medium was changed 1 time on day 3 of inoculation.

(3) Measurement of the balling rate: after 6 days of culture, non-adherent, clonally growing tumor spheres were obtained, with diameters ≥ 50.0 μm being defined as tumor spheres. Tumor spheres from each well were counted. The balling rate is the average number of tumor balls per well/total number of inoculated viable cells x 100%.

The results of the spherulization rate measurements (Table 2, FIG. 6) show that the spherulization rates of HUSSLCs after treatment with 100, 500, 1000-fold diluted 1-2F monoclonal cell supernatants were lower than those of HUSSLCs treated with SP2/0 cell supernatant (Cont), P <0.05, and the differences were statistically significant; compared with the supernatant of the 1-2F monoclonal cells diluted by 1000 times, the HUSSLCs treated by the supernatant of the 1-2F monoclonal cells diluted by 100 times have lower balling rate, P is less than 0.05, and the difference has statistical significance. Different dilution ratios of 1-2F monoclonal cell supernatants (1:1000, 1:500, 1: 100) reduced HUSSLCs spheronization rates in a concentration-dependent manner.

TABLE 2 comparison of the Balling Rate of HUSSLCs under the action of 1-2F monoclonal cell supernatants at different dilution ratios

Note:^*: the difference was statistically significant (P) compared to the supernatant of SP2/0 cells<0.05)；^#: the differences were statistically significant (P) compared to 1-2F monoclonal cell supernatants (1:1000)<0.05)。

Example 3 agar colony formation assay the Effect of monoclonal antibodies on the formation of HUSSLCs agar colonies

(1) The supernatant of SP2/0 cells and the supernatant of positive hybridoma cells (1:1000, 1:500, 1: 100) at different dilution ratios were exposed to HUSSLCs for 48 hours.

(2) Preparing agar: three distilled water was used to prepare 2 kinds of low melting point agarose liquids with concentration of 1.2% and 0.6%, which were autoclaved and kept in a dissolved state at 40 ℃. 20% FBS DMEM medium and 1.2% low melting point agarose liquid were mixed in equal proportions, the mixture was transferred to a 24-well plate at 0.5 ml/well and allowed to solidify as the bottom agar. 20% FBS DMEM medium and 0.6% low melting point agarose liquid were mixed in equal proportion, the mixture was transferred to a 24-well plate, 1000 HUSSLCs cells were added to each well, and mixed well as upper agar. After the upper layer agarose solution solidified, the medium was transferred to an incubator and cultured for 14 days, and 500. mu.l of DMEM medium containing 20% FBS was added every 5 days.

(3) Determination of colony formation rate: the number of cells ≧ 20 was defined as colony, and the number of colonies was counted using a fluorescence inverted microscope. Colony formation rate-average number of colonies per well/total number of viable cells inoculated x 100%.

The results of agar colony formation assay (Table 3, FIG. 7) show that HUSSLCs treated with 100, 500, 1000-fold diluted 1-2F monoclonal cell supernatants all had lower agar colony formation rates, P <0.05, and the differences were statistically significant, compared to HUSSLCs treated with SP2/0 cell supernatant (Cont); the agar colony formation rate of HUSSLCs after treatment with 100-fold diluted 1-2F monoclonal cell supernatants was lower, P <0.05, compared to 1000-fold diluted 1-2F monoclonal cell supernatants, with a statistically significant difference. Different dilution ratios of 1-2F monoclonal cell supernatants (1:1000, 1:500, 1: 100) reduced HUSSLCs spheronization rates in a concentration-dependent manner.

TABLE 3 comparison of HUSSLCs colony formation rates by supernatant from 1-2F monoclonal cells at different dilution ratios

Note:^*: the difference was statistically significant (P) compared to the supernatant of SP2/0 cells<0.05)；^#: the differences were statistically significant (P) compared to 1-2F monoclonal cell supernatants (1:1000)<0.05)。

Example 4 wound healing experiments to examine the Effect of monoclonal antibodies on HUSSLCs migration

(1) The supernatant of SP2/0 cells and the supernatant of positive hybridoma cells (1:1000, 1:500, 1: 100) at different dilution ratios were exposed to HUSSLCs for 48 hours.

(2) The treated HUSSLCs cells were seeded into 6-well cell culture plates and diluted to 5X 10 with 10% FBS-containing DMEM complete medium⁵Individual cells/well, photographs were taken when the cells fused up to 90%.

(3) Scratches were made by scribing tip across the center of the bottom of a 6-well plate. The debris and floating cells were then washed 2 times with PBS.

(4) After the scratch was made, the cells were cultured and after 24 hours, the same wound site was photographed and the cells in the wound area were counted. SP2/0 cell supernatant treated cells were used as a control group and relative cell mobilities were calculated.

The results of the wound healing test (table 4, fig. 8) show that the migration rates of HUSSLCs treated with 100, 500, 1000-fold diluted 1-2F monoclonal cell supernatants were all lower, P <0.05, compared to the HUSSLCs treated with SP2/0 cell supernatant (Cont), the differences being statistically significant; HUSSLCs treated with 100-fold diluted 1-2F monoclonal cell supernatants had lower mobility, P <0.05, and the differences were statistically significant compared to 1000-fold diluted 1-2F monoclonal cell supernatants. Different dilution ratios of 1-2F monoclonal cell supernatants (1:1000, 1:500, 1: 100) reduced HUSSLCs spheronization rates in a concentration-dependent manner.

TABLE 4 comparison of cell migration rates of HUSSLCs under the action of 1-2F monoclonal cell supernatants at different dilution ratios

Note:^*: the difference was statistically significant (P) compared to the supernatant of SP2/0 cells<0.05)；^#: the differences were statistically significant (P) compared to 1-2F monoclonal cell supernatants (1:1000)<0.05)。

Example 5 flow cytometry to examine the Effect of monoclonal antibodies on HUSSLCs CD133 expression

(1) The supernatant of SP2/0 cells and the supernatant of positive hybridoma cells (1:1000, 1:500, 1: 100) at different dilution ratios were exposed to HUSSLCs for 48 hours.

(2) Press 10⁵Cells/ml were seeded into William's E medium (containing 20% FBS) and incubated at room temperature for 15-30 minutes to block non-specific sites. Cells were washed 2 times with PBS and resuspended in 990. mu.l PBS. Then, 10. mu.l of antibody (including PE-CD133 antibody and isotype control PE-IgG) was added to the cell suspension. After incubation at 4 ℃ for 30min in the dark, cells were washed 2 times with PBS, fixed with 0.1% formaldehyde and FACS Calibur^TMAnd (5) detecting the system. All data are analyzed by Flow jo7.6.1 software, and images are output in a PDF format; and then using Adobe Photoshop to edit the image.

The results of the flow cytometry analysis of the PE-labeled CD133 antibody (table 5, fig. 9) showed that the percentage of CD133 positive cells was lower for HUSSLCs treated with 100, 500, 1000-fold diluted 1-2F monoclonal cell supernatants compared to the HUSSLCs treated with SP2/0 cell supernatant (Cont), with P <0.05, the difference being statistically significant; the percentage of CD133 positive cells was lower for HUSSLCs treated with 100, 500 fold diluted 1-2F monoclonal cell supernatants compared to 1000 fold diluted 1-2F monoclonal cell supernatants, P <0.05, the difference being statistically significant. The 1-2F monoclonal cell supernatants (1:1000, 1:500, 1: 100) at different dilution ratios reduced the percentage of HUSSLCs CD133 positive cells in a concentration-dependent manner.

TABLE 5 comparison of the percentage of HUSSLCs CD 133-positive cells under the action of 1-2F monoclonal cell supernatants at different dilution ratios

Note:^*: the difference was statistically significant (P) compared to the supernatant of SP2/0 cells<0.05)；^#: the differences were statistically significant (P) compared to 1-2F monoclonal cell supernatants (1:1000)<0.05)。

Example 6 Western blot assay to examine the Effect of monoclonal antibodies on the expression of HUSSLCs CD44 protein

(1) The supernatant of SP2/0 cells and the supernatant of positive hybridoma cells (1:1000, 1:500, 1: 100) at different dilution ratios were exposed to HUSSLCs for 48 hours.

(2) Preparing a whole-cell extract: cell lysis buffer was prepared for subsequent experiments: 150mM NaCl, 0.2% NP-40, 50mM Tris-HClpH7.4, 0.2mM EDTA, 0.1mM Na3VO4, 0.5mM4NPP, 0.5mM Na F, 1M beta-Me, 10% glycerol, 1. mu.g/ml aprotinin, 0.5. mu.g/ml zymoxalin peptide, protease inhibitor. After washing the cells with PBS, 1ml of a cell lysis enzyme buffer was added and incubated at 4 ℃ for half an hour. The cell lysate was collected, 13200g was centrifuged for 5 minutes, and the supernatant was collected.

(3) The Bradford assay measures the protein content of cell lysates (supernatants).

(4) 40. mu.g of the extracted protein was separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane.

(5) The membrane was blocked using 5% BSA at ambient temperature for 2 hours.

(6) The membrane was incubated overnight at 4 ℃ with mouse anti-human beta-actin monoclonal antibody and rabbit anti-human CD44 polyclonal antibody, respectively, as primary antibodies.

(7) The polyvinylidene fluoride membrane was washed with 1 × Tris and incubated with horseradish peroxidase secondary antibody for 2 hours at room temperature.

(8) Polyvinylidene fluoride membranes were washed with 1 × TBS and protein expression was detected with an enhanced chemiluminescence reagent.

(9) And (3) analyzing and calculating the ratio of the CD44 to the beta-actin protein strip gray scale by using image analysis software. The gray scale ratio of CD44 to β -actin protein bands after treatment of HUSSLCs with SP2/0 medium supernatant was defined as 1.00 and normalized to relative density.

(10) The above experiment was repeated 3 times, and the data of 3 independent experiments are expressed as mean ± standard deviation (n ═ 3).

The results of western blot analysis (table 6, fig. 10) show that the relative density of CD44 protein was lower for HUSSLCs after treatment with 100, 500-fold diluted 1-2F monoclonal cell supernatants compared to HUSSLCs treated with SP2/0 cell supernatant (Cont), P <0.05, the difference being statistically significant; the relative density of CD44 protein was lower for HUSSLCs treated with 100-fold diluted 1-2F monoclonal cell supernatants compared to 1000-fold diluted 1-2F monoclonal cell supernatants, P <0.05, with statistically significant differences. The 1-2F monoclonal cell supernatants (1:1000, 1:500, 1: 100) at different dilution ratios reduced the expression level of HUSSLCs CD44 protein in a concentration-dependent manner.

TABLE 6 comparison of HUSSLCs CD44 protein expression under the action of 1-2F monoclonal cell supernatants at different dilution ratios

Note:^*: the difference was statistically significant (P) compared to the supernatant of SP2/0 cells<0.05)；^#: the differences were statistically significant (P) compared to 1-2F monoclonal cell supernatants (1:1000)<0.05)。

Example 7 Western blot assay to examine the Effect of monoclonal antibodies on the expression of HUSSLCs ABCG2 protein

The rabbit anti-human CD44 polyclonal antibody of example 6 was replaced with the mouse anti-human ABCG2 monoclonal antibody, and the procedure was the same as in example 6.

The results of western blot analysis (table 7, fig. 11) show that the ABCG2 protein of HUSSLCs treated with 100, 500, 1000-fold diluted 1-2F monoclonal cell supernatants was lower in relative density, P <0.05, and the difference was statistically significant, compared to the HUSSLCs treated with SP2/0 cell supernatant (Cont); the relative density of ABCG2 protein was lower for HUSSLCs treated with 100-fold diluted 1-2F monoclonal cell supernatants compared to 1000-fold diluted 1-2F monoclonal cell supernatants, P <0.05, with statistically significant differences. The 1-2F monoclonal cell supernatants (1:1000, 1:500, 1: 100) at different dilution ratios reduced the expression level of HUSSLCs ABCG2 protein in a concentration-dependent manner.

TABLE 7 comparison of HUSSLCs ABCG2 protein expression under the action of 1-2F monoclonal cell supernatants at different dilution ratios

Note:^*: the difference was statistically significant (P) compared to the supernatant of SP2/0 cells<0.05)；^#: the differences were statistically significant (P) compared to 1-2F monoclonal cell supernatants (1:1000)<0.05)。

Example 8 Western blotting assay to examine the Effect of monoclonal antibodies on the expression of HUSSLCs Bmi1 protein

The rabbit anti-human CD44 polyclonal antibody of example 6 was replaced with rabbit anti-human Bmi1 monoclonal antibody, and the procedure was the same as in example 6.

The results of western blot analysis (table 8, fig. 12) show that relative density of Bmi1 protein was lower for HUSSLCs after treatment with 100, 500-fold diluted 1-2F monoclonal cell supernatants compared to HUSSLCs treated with SP2/0 cell supernatant (Cont), P <0.05, the difference being statistically significant; the relative density of Bmi1 protein was lower for HUSSLCs treated with 100-fold diluted 1-2F monoclonal cell supernatants compared to 1000-fold diluted 1-2F monoclonal cell supernatants, with P <0.05, with statistically significant differences. The 1-2F monoclonal cell supernatants (1:1000, 1:500, 1: 100) at different dilution ratios reduced the expression level of HUSSLCs Bmi1 protein in a concentration-dependent manner.

TABLE 8 comparison of HUSSLCs Bmi1 protein expression by 1-2F monoclonal cell supernatants at different dilution ratios

Note:^*: the difference was statistically significant (P) compared to the supernatant of SP2/0 cells<0.05)；^#: the differences were statistically significant (P) compared to 1-2F monoclonal cell supernatants (1:1000)<0.05)。

Example 9 Western blotting experiment to examine the Effect of monoclonal antibodies on the expression of HUSSLCs Nanog protein

The rabbit anti-human CD44 polyclonal antibody of example 6 was replaced with rabbit anti-human Nanog monoclonal antibody in the same manner as in example 6.

The results of Western blot analysis (Table 9, FIG. 13) show that HUSSLCs treated with 100, 500 fold diluted 1-2F monoclonal cell supernatants had lower relative densities of Nanog protein, P <0.05, and the differences were statistically significant, compared to HUSSLCs treated with SP2/0 cell supernatant (Cont); relative density of Nanog was lower for HUSSLCs treated with 100-fold diluted 1-2F monoclonal cell supernatants compared to 1000-fold diluted 1-2F monoclonal cell supernatants, P <0.05, and the difference was statistically significant. The 1-2F monoclonal cell supernatants (1:1000, 1:500, 1: 100) at different dilution ratios reduced the expression level of HUSSLCs Nanog protein in a concentration-dependent manner.

TABLE 9 comparison of HUSSLCs Nanog protein expression by supernatant from 1-2F monoclonal cells at different dilution ratios

Note:^*: the difference was statistically significant (P) compared to the supernatant of SP2/0 cells<0.05)；^#: the differences were statistically significant (P) compared to 1-2F monoclonal cell supernatants (1:1000)<0.05)。

Example 10 Western blot assay to examine the Effect of monoclonal antibodies on the expression of HUSSLCs Oct4 protein

The rabbit anti-human CD44 polyclonal antibody of example 6 was replaced with rabbit anti-human Oct4 monoclonal antibody, and the procedure was the same as in example 6.

The results of western blot analysis (table 10, fig. 14) show that HUSSLCs treated with 100, 500-fold diluted 1-2F monoclonal cell supernatant had lower Oct4 protein relative densities, P <0.05, and the differences were statistically significant, compared to HUSSLCs treated with SP2/0 cell supernatant (Cont); relative density of Oct4 protein was lower for HUSSLCs treated with 100-fold diluted 1-2F monoclonal cell supernatants compared to 1000-fold diluted 1-2F monoclonal cell supernatants, P <0.05, with statistically significant differences. The 1-2F monoclonal cell supernatants (1:1000, 1:500, 1: 100) at different dilution ratios reduced the expression level of HUSSLCs Oct4 protein in a concentration-dependent manner.

TABLE 10 comparison of HUSSLCs Oct4 protein expression by supernatant from 1-2F monoclonal cells at different dilution ratios

Note:^*: the difference was statistically significant (P) compared to the supernatant of SP2/0 cells<0.05)；^#: the differences were statistically significant (P) compared to 1-2F monoclonal cell supernatants (1:1000)<0.05)。

Example 11 Western blot assay to examine the Effect of monoclonal antibodies on the expression of HUSSLCs ALDH1 protein

The rabbit anti-human CD44 polyclonal antibody of example 6 was replaced with rabbit anti-human ALDH1 monoclonal antibody, and the procedure was the same as in example 6.

The results of western blot analysis (table 11, fig. 15) show that the relative density of ALDH1 protein was lower for HUSSLCs treated with 100, 500, 1000-fold diluted 1-2F monoclonal cell supernatants compared to HUSSLCs treated with SP2/0 cell supernatant (Cont), P <0.05, the difference being statistically significant; the relative density of ALDH1 protein was lower in HUSSLCs treated with 100-500 fold diluted 1-2F monoclonal cell supernatants compared to 1000 fold diluted 1-2F monoclonal cell supernatants, with P <0.05, with statistical differences. The 1-2F monoclonal cell supernatants (1:1000, 1:500, 1: 100) at different dilution ratios reduced the expression level of HUSSLCs ALDH1 protein in a concentration-dependent manner.

TABLE 11 comparison of HUSSLCs ALDH1 protein expression by supernatant from 1-2F monoclonal cells at different dilution ratios

Note:^*: the difference was statistically significant (P) compared to the supernatant of SP2/0 cells<0.05)；^#: the differences were statistically significant (P) compared to 1-2F monoclonal cell supernatants (1:1000)<0.05)。

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (7)

1. The hybridoma cell strain is characterized in that the preservation number is CCTCC No. C2018182.

2. The monoclonal antibody is produced by a hybridoma cell line with the preservation number of CCTCC No. C2018182.

3. Use of the monoclonal antibody of claim 2 for the preparation of a medicament and/or formulation for inhibiting self-renewal, unlimited proliferation, anchorage-independent growth, tumorigenicity, migration or invasion of stem-like cells of human uterine leiomyosarcoma.

4. Use of the monoclonal antibody of claim 2 for the preparation of a medicament and/or formulation for inhibiting the expression of the human uterine leiomyosarcoma stem cell-like cell marker CD133 protein.

5. Use of a monoclonal antibody according to claim 2 for the preparation of a medicament and/or a formulation for the treatment of tumors; the tumor is human uterine leiomyosarcoma.

6. A medicament comprising the monoclonal antibody of claim 2 and a pharmaceutically acceptable excipient.

7. A kit comprising the monoclonal antibody of claim 2.

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