CN100398562C - Anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUF10 and its application - Google Patents

Abstract

The invention provides an anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUF10. Using human bone marrow mesenchymal stem cells as an antigen, a monoclonal antibody against human mesenchymal stem cells was prepared by using hybridoma technology, and the monoclonal antibody was used as a probe to perform flow cytometry, immunohistochemistry, and immunofluorescence staining. 1. A method for detecting mesenchymal stem cells by immunoblotting. The invention provides an effective tool for studying the specific markers of the mesenchymal stem cells, and also provides a platform for further clarifying the biological characteristics of the mesenchymal stem cells, and can be popularized and applied to various detection techniques and clinical research.

Description

Anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUF10 and its application

Technical field:

The invention belongs to the field of biological technology, and relates to an anti-human bone marrow mesenchymal stem cell monoclonal antibody and its application.

Background technique:

Mesenchymal Stem Cells (Mesenchymal Stem Cells, Mesenchymal Stem Cells) is a hotspot in the field of adult stem cell research in recent years, which can be isolated from bone marrow, umbilical cord blood, adipose tissue, synovium, skeletal muscle, lung, and deciduous teeth. Potential to divide, proliferate, and differentiate into various cells. Under certain in vivo or in vitro induction conditions, it can differentiate into cells of various germ layers, such as adipocytes, chondrocytes, osteoblasts, tenocytes, muscle cells, and astrocytes. Cells, neuron cells, epithelial cells, etc.; mesenchymal stem cells have weak immunogenicity, do not express MHC-II molecules and T cell co-stimulatory molecules, and have immunomodulatory activity, which makes the application of allogeneic mesenchymal stem cells a Possibly; through genetic modification methods, mesenchymal stem cells can be directed to express some specific molecules as an ideal carrier for cell-mediated gene therapy. Mesenchymal stem cells are the seed cells of tissue engineering and regenerative medicine, and have broad prospects in clinical application. At present, many research centers in the United States and Europe have carried out phase I and II clinical trials of autologous and allogeneic mesenchymal stem cells in the treatment of various diseases, mainly used in myocardial infarction, osteogenesis imperfecta, large bone defects, cartilage defects, tendon Injury, repair and reconstruction of ligament damage, and treatment of Metachromatic Leukodystrophy (MLD), Hurler Syndrome (MPSIH), severe aplastic anemia, graft-versus-host disease and other diseases The treatment has a certain clinical effect; China is also embarking on phase I clinical research on the treatment of major diseases with mesenchymal stem cells.

Bone marrow mesenchymal stem cells were first confirmed by Friedenstein in the 1960s, and they are a kind of non-hematopoietic stem cells in the bone marrow. Bone marrow mesenchymal stem cells exist in low density in the bone marrow, about 10 ⁴ to 10 ⁵ mononuclear cells contain one mesenchymal stem cell, but they can be cultured in vitro for 50 times without changing their phenotypic characteristics and differentiation potential . So far, mesenchymal stem cells have no specific surface markers. They do not express CD34, CD45, CD14, glycophorin A, and T or B lymphocyte surface markers. It is believed that the relative specific surface antigens include SH2, SH3, SH4, STRO -1, CD166, etc. At present, various laboratories mainly obtain mesenchymal stem cells through density gradient centrifugation combined with adherent culture method and immunological sorting method for positive or negative screening. The cultured and passaged mesenchymal stem cells are a group of relatively uniform cells, but different isolation and culture conditions The obtained mesenchymal stem cells may come from different industry groups, and there may be certain differences in their biological characteristics. Researchers are working hard to find specific markers of mesenchymal stem cells to further understand their biological characteristics.

Since the 1990s, researchers have developed a variety of monoclonal antibodies related to human mesenchymal stem cells through hybridoma technology: in 1991, Paul J.Simmons et al. obtained new human bone marrow stromal cells by immunizing mice with bone marrow CD34 ⁺ cells. Monoclonal antibody STRO-1; In 1992, Haynesworth SE et al. developed three monoclonal antibodies SH2, SH3, and SH4 for mesenchymal stem cells. Further research later confirmed that the epitope of SH2 is located on CD105, and the epitope of SH3 and SH4 Located on CD73; in 1997, the monoclonal antibody HOP-26 developed by JC Joyner et al. was specific for the precursor cells of osteogenic differentiation of mesenchymal stem cells, and the study confirmed that its antigenic epitope was a peptide on CD63; The osteoblast cells induced and differentiated from bone marrow mesenchymal stem cells were immunized with mice to obtain monoclonal antibody SB-10, which is related to the osteogenic differentiation of mesenchymal stem cells. The epitope of SB-10 is located on CD166; Hyun-Jung Hoo in 2005 et al. reported that mice were immunized with bone marrow mesenchymal stem cells and cell membrane protein extracts, and monoclonal antibodies YS08, YS14, and YS18 were obtained. The research on monoclonal antibodies of mesenchymal stem cells and related cells has enriched the understanding of the surface molecules of mesenchymal stem cells and their differentiation antigens, but there is still a lack of comprehensive understanding of the phenotype of mesenchymal stem cells, and no single antibody has been found. Therefore, further understanding of the specific markers and biological characteristics of mesenchymal stem cells will advance the research and application of mesenchymal stem cells.

Contents of the invention

One object of the present invention is to provide anti-human bone marrow mesenchymal stem cell monoclonal antibody, the subtype of the monoclonal antibody is IgG1, κ type, named ZUF10, which can specifically bind to the surface molecules of human bone marrow mesenchymal stem cells, and can be passed through ZUF10 Western blotting was used to detect the extracted human bone marrow mesenchymal stem cell proteins, and a single specific positive band could be obtained. The monoclonal antibody has no cross-reaction with human peripheral blood lymphocytes, human blood system cell lines and other tissue cell lines, human mesenchymal tissue and non-mesenchymal tissue, rat mesenchymal stem cells, and other animal bone marrow cells.

The second object of the present invention is to provide a method for preparing monoclonal antibodies against bone marrow human mesenchymal stem cells, which is achieved through the following technical solutions:

(1) Preparation of immunogen: The third to fifth generation human bone marrow mesenchymal stem cells cultured after subculture from multiple donors and cryopreserved and revived were used as antigens for animal immunization. Human bone marrow mesenchymal stem cells were isolated and cultured by Ficoll-paque density gradient centrifugation combined with adherence screening method. The currently recognized human mesenchymal stem cell surface molecules CD29, CD44, CD166, CD105 (SH2) positive markers showed a single peak, and hematopoietic cell differentiation The expression of antigen CD14, CD34, CD45, HLA-DR is negative, and it is a highly homogeneous cell population; it can be passaged for many times, and the number of cells expanded to the 8th passage can reach about (2~3)× ¹⁰¹⁰ ; through in vitro directional induction Can differentiate into osteoblasts and adipocytes of mesenchymal origin, and can also differentiate into cells of other germ layer origin (such as neuron-like cells); the cryopreservation method used has a high survival rate of human bone marrow mesenchymal stem cells after thawing , does not affect the growth characteristics, immunophenotype, and proliferation and multilineage differentiation potential of cells.

(2) Animal immunization: Select 2-3 mixed human bone marrow mesenchymal stem cells from donors and immunize BALB/c mice in order to produce specific monoclonal antibodies against the common recognition site of mesenchymal stem cells. Each mouse was injected intraperitoneally with 1.0×10 ⁶ cells, once a week, 3 times in total; booster immunization was given once in the 4th week, and fusion was performed 3 days later.

(3) Establishment of hybridoma cell lines: Splenocytes from immunized mice were fused with mouse myeloma cells (SP2/0) mediated by PEG. The fused cells were selectively cultured in HAT medium, and positive clones were screened by indirect immunofluorescence. The hybridoma cells in positive wells were cloned and cultured by limiting dilution until the positive rate of cloned cell antibody was 100%. The hybridoma cells were continuously passaged in vitro for more than 3 months and repeatedly frozen and revived until the cell lines can stably secrete monoclonal antibodies.

(4) Preparation and purification of monoclonal antibody: The established hybridoma cells were injected into the peritoneal cavity of mice pretreated with paraffin oil to induce ascites. The induced ascites was purified by salting out and ion exchange.

The third object of the present invention is to provide hybridoma cells producing monoclonal antibodies, which are fused, screened, cloned, passaged and repeatedly frozen by immunized BALB/c mouse splenocytes and mouse myeloma SP2/0 cells 1. The mouse hybridoma cell line ZUF10 obtained after resuscitation can stably secrete the monoclonal antibody ZUF10, which was preserved in the China Center for Type Culture Collection with the preservation number: CCTCC No.C200513 and the preservation date: August 30, 2005 .

The fourth object of the present invention is to establish a method for detecting mesenchymal stem cells by using the monoclonal antibody through techniques such as flow cytometry, immunohistochemistry, immunofluorescent staining, and western blotting.

Using the anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUF10 as a probe, the cultured and expanded mesenchymal stem cells and the mesenchymal stem cells in the bone marrow cell suspension can be detected and quantitatively analyzed by flow cytometry.

Using anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUF10, immunohistochemical staining was used to detect the expression of corresponding antigens of mesenchymal stem cells in cultured and expanded mesenchymal stem cells, bone marrow tissue slices and other tissue slices, and the expression of mesenchymal stem cells in the mesenchymal stem cells The distribution of mesenchymal stem cells was analyzed for localization.

Using anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUF10, combined with fluorescent secondary antibody, immunofluorescent staining was used to label the corresponding protein molecules of cultured and expanded mesenchymal stem cells, whole bone marrow cells, other cells and frozen sections of tissues. The expression of corresponding protein molecules of mesenchymal stem cells is localized and quantitatively analyzed, and the distribution of mesenchymal stem cells in tissues can be detected.

The anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUF10 was used to detect the expression of the corresponding antigen of the monoclonal antibody ZUF10 in cultured and expanded mesenchymal stem cells and other related cells.

The present invention applies the established culture and cryopreservation system of human bone marrow mesenchymal stem cells, adopts bone marrow mesenchymal stem cells from multiple donors as the antigen to immunize BALB/c mice, and successfully prepares a new anti-human bone marrow mesenchymal stem cell Stem cell monoclonal antibody, which can be used for the identification of mesenchymal stem cells, provides an effective tool for studying the specific markers of mesenchymal stem cells, and also provides a platform for further elucidating the biological characteristics of mesenchymal stem cells, and can be extended Used in a variety of detection techniques and clinical research.

Description of drawings:

Figure 1 shows human bone marrow mesenchymal stem cells obtained by density gradient centrifugation and adherent culture. A is the cultured primary human bone marrow mesenchymal stem cells, B is the cultured human bone marrow mesenchymal stem cells after cryopreservation and thawing.

Fig. 2 is the growth curve of cultured and expanded first-generation and fourth-generation human bone marrow mesenchymal stem cells.

Figure 3: The growth curve of the fourth generation human bone marrow mesenchymal stem cells before and after cryopreservation.

Figure 4: Cultured fourth-generation human bone marrow mesenchymal stem cells were labeled with CD14-FITC, CD45-FITC, CD44-FITC, HLA-DR-FITC, CD34-PE, CD29-PE, CD166-PE, CD105-PE , the picture shows flow cytometry analysis.

Figure 5: Human bone marrow mesenchymal stem cells are induced to differentiate into osteoblasts. A is induced osteoblast differentiation, B is Von Kossa staining after induction of osteogenic differentiation.

Figure 6: Human bone marrow mesenchymal stem cells are induced to differentiate into fat. A is induced differentiation of adipocytes, B is oil red O staining after induction of adipose differentiation.

Figure 7: Directed differentiation of human bone marrow mesenchymal stem cells into neuron-like cells. A is induced differentiation of neuron-like cells, B, C, D, E are the detection of neural stem cell marker Nestin, neuron marker NSE, neuron marker NF-M, Expression of the astrocyte marker GFAP.

Figure 8: Detection of human bone marrow mesenchymal stem cells by indirect immunofluorescence staining with monoclonal antibody ZUF10.

Figure 9: The expression of the corresponding antigen of anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUF10 in bone marrow tissue was detected by immunohistochemical staining.

Figure 10: Detection of human bone marrow mesenchymal stem cells by flow cytometry with monoclonal antibody ZUF10. A is the negative control, B is the result of flow cytometry analysis with ZUF10 as the probe.

Figure 11 Detects human bone marrow mesenchymal stem cells by immunohistochemical staining with monoclonal antibody ZUF10. A is a slide of human bone marrow mesenchymal stem cells, and B is a mass of human bone marrow mesenchymal stem cells.

FIG. 12 : The expression of the corresponding antigen in human bone marrow mesenchymal stem cells was detected by Western blotting with the monoclonal antibody ZUF10.

Detailed ways

Example 1: Isolation, culture, identification, cryopreservation and recovery of human bone marrow mesenchymal stem cells

(1) Isolation and culture of human bone marrow mesenchymal stem cells

The bone marrow of six healthy donors was collected under sterile conditions, anticoagulated with heparin, mononuclear cells were collected by density gradient centrifugation with Ficoll-paque (specific gravity 1.077), seeded at a density of 4×10 ⁵ cells/cm ² , and contained 10% (V/V) Low-sugar DMEM (LG-DMED) culture solution of fetal bovine serum was placed in an incubator at 37° C. with a volume fraction of 5% CO ₂ . Replace the culture medium after 48 hours, discard the non-adherent cells, and there are spindle-shaped adherent cells, and then change the medium every 3 days, and the primary culture cells adhere to and fuse to 80% to 90% in 15 to 20 days, and the arrangement has obvious directionality , into a swirl, mesh, radial shape, similar to fibroblasts (see Figure 1), digested with 0.25% trypsin-1mmol EDTA, subcultured in bottles at a ratio of 1:3, and labeled as P1 cells. During the subculture process, the medium was changed every 3 days. After the cells reached 90% confluence, they were digested and passaged, and marked as P2, and so on.

(2) Identification of growth characteristics of human bone marrow mesenchymal stem cells

The cultured and passaged human bone marrow mesenchymal stem cells of the first and fourth passages were seeded in a 24-well culture plate at a density of 2× ¹⁰⁴ cells/well, and cultured for 1, 2, 3, 4, 5, 6 , 7, and 8 days for cell kinetic analysis. The culture has the following common characteristics: After the cells are subcultured, the cells will completely adhere to the wall within 12 hours, and the cell shape will become a spindle cell again. The subculture incubation period is about 24-36 hours; After the end of the proliferative phase, it enters the plateau phase; there is no significant difference in the growth of the cells of the fourth generation and the first generation (see Figure 2).

(3) Phenotype identification of human bone marrow mesenchymal stem cells

The cells of the first, third and fifth generations were cultured and passaged, and CD14-FITC, CD45-FITC, CD44-FITC, HLA-DR-FITC, CD34-PE, CD29-PE, CD166-PE, CD105-PE monoclonal antibodies were used as Probes, Expression of Mesenchymal Stem Cell Surface Molecules Analyzed by Flow Cytometry. The results showed that CD29, CD44, CD166, and CD105 (SH2) positive markers appeared single peaks, and the expression of hematopoietic cell differentiation antigens CD14, CD34, CD45, and HLA-DR was negative (see Figure 4). There was no significant difference in the expression of surface markers of the mesenchymal stem cells of different passages after passage and expansion (see Table 1), indicating that the cultured and expanded human bone marrow-derived mesenchymal stem cells are a homogeneous cell population.

Table 1 Immunophenotype of adult bone marrow mesenchymal stem cells (%, x±s) (n=6)

(4) Identification of multi-differentiation potential of human bone marrow mesenchymal stem cells

When nearly 70% of the subcultured cells are confluent, replace the culture medium, add osteogenesis induction medium (LG-DMEM, 10% fetal bovine serum, 10 ^-8 M dexamethasone, 10mM β-glycerol phosphate, 0.25mM ascorbic acid), Adipose induction medium, neural differentiation pre-induction medium (LG-DMEM, 20% fetal bovine serum, 1 mM thioglycerol).

After 3-4 days of osteogenic induction culture, about 30% of the cells changed in shape, from the original spindle type to cubic or polygonal, and calcified spots appeared after 7 days. With the prolongation of induction time, the cubic or polygonal cells and Calcified plaques increased significantly, and the cells formed extensive and uniform mineralized nodular structures at about 14 to 21 days. After 21 days of culture, staining by Von Kossa method showed brown-black extracellular matrix calcium salt deposits (see Figure 5), and the control group had no ability to form mineralized nodules.

After adipogenic induction and culture for 1 week, the cell morphology began to become round or polygonal, and the cells were arranged disorderly. It was observed that small lipid droplets with high refractive index appeared in the cytoplasm, and gradually aggregated into large lipid droplets with the prolongation of induction time. lipid droplets. After 21 days of culture, oil red O staining, the lipid droplets were orange red, and more than 80% of the mesenchymal stem cells were induced into adipocytes (see FIG. 6 ).

After 24 hours of neural differentiation pre-induction, the cell morphology did not change significantly. When replaced with serum-free LG-DMEM containing 5mM thioglycerol, most of the mesenchymal stem cells became typical neuron-like cells in 3-5h after induction. Both simple bi-level cells and complex multi-level cells are present, and multiple neuron-like cell processes can extend each other and form a network (see Figure 7). Immunohistochemical staining analysis showed that the cell bodies and some processes of neuron-like cells were positive for the expression of neural stem cell markers Nestin, neuron marker NSE, and neuron marker NF-M, and the expression of astrocyte marker GFAP was negative (see Fig. 7).

(5) Cryopreservation and recovery of human bone marrow mesenchymal stem cells

After digesting the cultured mesenchymal stem cells, resuspend the cells in cryopreservation solution (LG-DMEM, 5% DMSO, 30% fetal calf serum), the final cell concentration is 1×10 ⁶ /ml, and use the programmed cryopreservation method Freezing, computer setting cooling program: drop from 4°C to 0°C at a rate of -1°C/min, equilibrate for 5 minutes, drop to -30°C at a rate of -1°C/min, and then drop to -5°C/min -80°C, quickly transferred to -196°C liquid nitrogen tank for storage. Take the cells out of liquid nitrogen and put them into a constant temperature water bath at 40°C to thaw quickly. The thawing is completed within 1 min. After the cells are recovered, the cells are counted by trypan blue staining. The recovery rate of trypan blue rejection is 87.67±2.52%. After 12 hours, the cells were completely adhered to the wall, and the cell morphology became spindle cells again. The cells begin to proliferate rapidly after a 2-3d dormant period, and the cells can reach 80% to 90% fusion in about 7 to 10 days. The cells are arranged in a swirl, mesh, and radial shape, which is basically the same as the shape of the cells before cryopreservation (see Figure 1 ). Compare the fourth-generation cells before and after cryopreservation, the growth characteristics of the cells (the cell growth curve was measured by MTT method, see Figure 3), the immune phenotype (see Table 2), and the induction differentiation potential (osteoblasts, adipocytes, nerve cells, etc.) Yuan-like cells) did not change.

Table 2 Cell immunophenotype before and after cryopreservation of the fourth generation human bone marrow mesenchymal stem cells (%, x±s) (n=6)

Example 2: Preparation and purification of anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUF10

(1) Animal immunity

Female BALB/c mice aged 6-8 weeks were used for the first immunization with 2 parts of mixed donor-derived mesenchymal stem cells of the third to fifth passages suspended in PBS, and about 1.0×10 ⁶ /mouse was injected into the peritoneal cavity of the mice. Immunization was continued on the 8th and 15th day, and the immunization method was the same as the first time. On the 20th day, blood was collected from the eyeballs of the mice after three immunizations, and the serum was separated by centrifugation. The serum was co-incubated with the cultured human mesenchymal stem cells to detect the serum antibody titer by indirect immunofluorescence staining, and the mice with high titers were selected for fusion. A booster immunization was carried out 3 days before fusion, and the number of cells was 2.0×10 ⁶ per mouse.

(2) Preparation of hybridoma cell lines

After completing the immunization process, spleen cells were prepared to be fused with mouse myeloma cells (SP2/0), and feeder cells were prepared the day before the fusion. Spleen cells were aseptically collected from mice during fusion, mixed with SP2/0 cells at a ratio of 10:1, fused with 50% PEG, resuspended in HAT selective medium after centrifugation, and inoculated on 96 plates containing feeder cells Cultured in a 37°C, 5% CO2 incubator, half of HAT culture medium was replaced on the 3rd, 5th, and 7th days after fusion, and HT culture medium was replaced two weeks later.

Observe the growth of hybridoma cells, wait for the clones to grow to 1/3-1/2 of the bottom area of the well, take the culture supernatant and incubate with mesenchymal stem cells, use Ig(G+M)-FITC fluorescent secondary antibody to indirect Antibody detection was performed by immunofluorescence staining, and positive clones were screened. The hybridoma cells in positive wells were cloned and cultured by limiting dilution until the positive rate of cloned cell antibody was 100%, at which time the positive cloned cells could be further expanded and cultured. The hybridoma cells were continuously passaged in vitro for more than 3 months and repeatedly frozen and revived, and the supernatant was collected regularly, and the antibody in the supernatant was detected by the method of screening antibodies until the cell line could stably secrete monoclonal antibodies.

(3) Preparation and purification of monoclonal antibodies

Adult BALB/c mice were selected and injected intraperitoneally with 0.5ml of liquid paraffin. After 1-2 weeks, monoclonal hybridoma cells in good growth state were collected, and each mouse was injected intraperitoneally with 0.5-1.0×10 ⁵ cell suspension. About 14 days after the inoculation of cells, the abdominal cavity of the mouse was obviously enlarged, and the abdominal cavity was opened after being killed by cervical dislocation, and the ascites was taken out. After centrifugation, the supernatant was collected, aliquoted, and stored at -20°C. Take 10ml of ascites antibody, add 10ml of normal saline to dilute, use salting-out method to crudely extract immunoglobulin with 50% ammonium sulfate, and then use DEAE-cellulose ion-exchange column to purify.

(4) Anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUF10 ascites titer and subtype identification

Take mouse ascites antibody, after doubling dilution, incubate with immunized mesenchymal stem cells, and detect with indirect immunofluorescence staining method (the method is the same as antibody screening), the anti-human bone marrow mesenchymal stem cell monoclonal antibody titer is 1:10 ⁶ . Determination of antibody subtypes The culture supernatant of hybridoma cells was taken, and detected by gold immunochromatography, which belonged to IgG1 type (light chain was κ type).

Example 3: Identification of the specificity of the monoclonal antibody ZUF10

(1) Identification of the specificity of monoclonal antibody ZUF10 in human blood system cells and human cell lines

Cell material: cultured human bone marrow mesenchymal stem cells, peripheral blood cells, human peripheral blood mononuclear cells separated by Ficoll-paque (specific gravity 1.077), whole bone marrow cells, bone marrow mononuclear cells separated by Ficoll-paque (specific gravity 1.077), HL -60 (acute myeloid leukemia cell line), NB4 (acute promyelocytic leukemia cell line), K562 (chronic myelogenous leukemia blast phase cell line), U-937 (monocytic macrophage cell line from histiocytic lymphoma) Cell differentiation cell line), HEL (acute erythroleukemia cell line), Jurkat (acute T lymphocytic leukemia cell line), Raji (Burkitt lymphoma cell line, B lymphocyte), KM3 (multiple myeloma cell line). Live cell indirect immunofluorescence staining was used to detect the expression of mesenchymal stem cell membrane antigen, and human bone marrow mesenchymal stem cells were used as a positive control (see Figure 8). Scattered positive cells could be seen in whole bone marrow cells and bone marrow mononuclear cells, and the rest cells are negative. The specific binding of monoclonal antibody ZUF10 to bone marrow mesenchymal stem cells can be used to distinguish mesenchymal stem cells and hematopoietic cells in bone marrow, and to identify bone marrow mesenchymal stem cells.

(2) Identification of the specificity of monoclonal antibody ZUF10 in human mesenchymal tissue and non-mesenchymal tissue

Tissue materials: tendons, ligaments, muscles, blood vessel walls, nerves, gallbladder, skin, lungs, liver, small intestine, fat, bone marrow, the above tissue samples are all from surgical specimens. Immunohistochemical staining was used for detection, and the paraffin section of human bone marrow mesenchymal stem cell aggregates was used as a positive control. Scattered positive cells could be seen in the bone marrow tissue (see FIG. 9 ), and the rest of the tissues were negative.

(3) Specific identification of monoclonal antibody ZUF10 in rat, mouse, dog, rabbit, chicken bone marrow cells and rat mesenchymal stem cells

Cell material: Rat, mouse, dog, rabbit, chicken whole bone marrow cells and cultured rat mesenchymal stem cells. Indirect immunofluorescence staining was used to detect human bone marrow mesenchymal stem cells as a positive control. The results showed that rat, mouse, dog, rabbit, chicken whole bone marrow cells and cultured rat mesenchymal stem cells were all negative. It shows that the monoclonal antibody ZUF10 is specific to human mesenchymal stem cells.

Example 4: Application of monoclonal antibody ZUF10 in detection of mesenchymal stem cells by flow cytometry

Human bone marrow mesenchymal stem cells cultured in vitro were collected, and the expression of surface molecules of mesenchymal stem cells was analyzed by indirect flow cytometry with monoclonal antibody ZUF10 as a probe. The results showed an obvious positive single peak (see Figure 10), the expression of molecules on the surface of mesenchymal stem cells was 82.17%-90.67%, and there was no significant difference in the expression of ZUF10 corresponding antigens on subcultured mesenchymal stem cells. The above results show that the monoclonal antibody ZUF10 has a high specific binding to the surface molecules of mesenchymal stem cells, which can be used for the detection and identification of mesenchymal stem cells, and can quantitatively detect the number of mesenchymal stem cells. Using monoclonal antibody ZUF10 as a probe to detect the number of bone marrow mesenchymal stem cells by flow cytometry can be used to study the characteristics and clinical diagnosis of fresh mesenchymal stem cells in bone marrow.

Example 5: Application of Monoclonal Antibody ZUF10 in Detection of Mesenchymal Stem Cells by Immunohistochemical Staining

The slices of human mesenchymal stem cells cultured in vitro were fixed, and the expression of the corresponding antigen of mesenchymal stem cells was detected by immunohistochemical staining with monoclonal antibody ZUF10. The results showed that more than 95% of the cells were positive (see Figure 11). Similarly, ZUF10 was used to detect the expression of corresponding antigens in paraffin sections of mesenchymal stem cell aggregates and bone marrow tissue by immunohistochemical staining. The results showed that more than 95% of the cells in the cell aggregates were positive, and there were scattered positive cells in the bone marrow tissue. (See Figure 9). The monoclonal antibody ZUF10 is used in the detection method of immunohistochemical staining, which can be used as a marker of mesenchymal stem cells, and can be used for localization and quantitative analysis of tissue mesenchymal stem cells.

Example 6: Application of Monoclonal Antibody ZUF10 in Detection of Mesenchymal Stem Cells by Immunofluorescent Staining

Using monoclonal antibody ZUF10 as a probe, combined with FITC-labeled fluorescent secondary antibody, the indirect immunofluorescence staining method was used to detect the mesenchymal stem cells cultured in vitro, and the results showed that 95% to 100% of the mesenchymal stem cells were positive (see Figure 8 ). The monoclonal antibody ZUF10 can be effectively applied to immunofluorescence staining, and the expression of the corresponding antigen in mesenchymal stem cells can be effectively observed through fluorescence microscope, laser confocal microscope and other tools, and the expression of the antigen in mesenchymal stem cells can be further studied. Biological properties. In addition, the monoclonal antibody ZUF10 can be used to detect mesenchymal stem cells in bone marrow cells and other tissues by indirect immunofluorescence staining.

Example 7: Using the monoclonal antibody ZUF10 to detect the expression of the corresponding antigen by Western blotting

The total protein of the mesenchymal stem cells cultured in vitro was extracted, and the expression of the corresponding antigen was detected by conventional Western blotting with the monoclonal antibody ZUF10, and a specific positive band was seen (see FIG. 12 ). The expression of the corresponding antigen of the monoclonal antibody ZUF10 in mesenchymal stem cells and related cells can be further studied by immunoblotting, as well as the biological characteristics of the antigen.

Claims (7)

1. monoclonal antibody ZUF 10 of human bone marrow mesenchymal stem cells, this monoclonal antibody hypotype is IgG1, the κ type, can combine with human marrow mesenchymal stem cell surface molecular specificity, the hybridoma that produces this monoclonal antibody is through merging by the BALB/c mouse splenocyte of immunity and mouse myeloma SP2/0 cell, screening, the clone, go down to posterity with frozen repeatedly, the mouse hybridoma cell that the recovery back obtains is ZUF10, energy stably excreting monoclonal antibody ZUF10, it is in China's typical culture collection center preservation, preserving number is: CCTCCNo.C200513, preservation date is: on August 30th, 2005.

2. the preparation method of monoclonal antibody ZUF 10 of human bone marrow mesenchymal stem cells according to claim 1 is characterized in that being achieved through the following technical solutions:

The hybridoma of claim 1 preservation is expelled to through the pretreated mouse peritoneal of paraffin oil, induces ascites, the ascites that induces obtains the monoclonal antibody of purifying with salting-out process and ion exchange method.

3. monoclonal antibody ZUF 10 of human bone marrow mesenchymal stem cells according to claim 1 is used in the vitro detection human mesenchymal stem cell.

4. application according to claim 3 is characterized in that: with monoclonal antibody ZUF10 is probe, cultivates the mescenchymal stem cell of amplification and the mescenchymal stem cell in the bone marrow cell suspension by Flow cytometry, and carries out quantitative analysis.

5. application according to claim 3, it is characterized in that: use monoclonal antibody ZUF10, detect the expression of the mescenchymal stem cell corresponding antigens in mescenchymal stem cell, myeloid tissue section and other tissue slicies of cultivating amplification by immunohistochemistry staining method, and the distribution of mescenchymal stem cell is positioned analysis.

6. application according to claim 3, it is characterized in that: with monoclonal antibody ZUF10 is probe, combined with fluorescent two is anti-, cultivate the corresponding antigens of the mescenchymal stem cell of amplification, full medullary cell, other cells and tissue freezing section with the immunofluorescence staining mark, expression to the corresponding antigens of mescenchymal stem cell positions and quantitative analysis, and can detect the distribution of mescenchymal stem cell in tissue.

7. application according to claim 3 is characterized in that: use monoclonal antibody ZUF10, detect its corresponding antigens in the mescenchymal stem cell of cultivating amplification and the expression in other relevant cells by immunoblotting.

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