CN111019898B - Human malignant phyllodes tumor cell line HJP-0320 and its application - Google Patents

Abstract

The invention discloses a human malignant foliar tumor cell line HJP-0320, which is preserved in China center for type culture Collection with the following preservation addresses: china, university of Wuhan, and preservation number is C2018233. The invention also discloses the application of the human malignant foliar tumor cell line HJP-0320 as a cell model for researching the tumorigenesis and development mechanism in the screening of antitumor drugs. The human foliar tumor cell line HJP-0320 is established from a Chinese human source, has short establishment time and stable biological hereditary property, is lack of a human malignant foliar tumor cell line on the market at present, and has great help for understanding the pathogenesis of Chinese foliar malignant tumor patients by taking the foliar tumor cell line as a research model.

Description

Human malignant phyllodes tumor cell line HJP-0320 and its application

technical field

The invention relates to the technical field of tumor cell lines, in particular to a human malignant phyllodes tumor cell line HJP-0320 and its application.

Background technique

Phylodes tumor of the breast is a rare breast tumor, accounting for about 1% of breast tumors. This tumor grows rapidly and often presents as a huge mass; it can be divided into benign, borderline, and malignant histologically; even benign phyllodes tumors can recur, and malignant phyllodes tumors can also undergo hematogenous metastasis. Chemotherapy and radiotherapy have uncertain therapeutic effects on phyllodes tumors. The current treatment that can reduce the chances of recurrence and metastasis is extended surgical resection, but even with extended surgical resection, the local recurrence rate of phyllodes tumors is still as high as 8-36%, and the rate of hematogenous metastasis of malignant phyllodes tumors is as high as 22%.

Currently, the mechanisms driving the malignant transformation of phyllodes tumors are still unclear. Existing molecular markers are also of limited value in predicting the biological behavior of tumors. Therefore, it is very necessary to strengthen the research on the malignant progression mechanism of phyllodes tumor cells, which is of great significance to curb the malignant progression of phyllodes tumors, reduce local recurrence, distant metastasis and the mortality of malignant phyllodes tumors. The establishment of reliable phyllodes tumor cell lines is a top priority.

Contents of the invention

Based on the above problems, the purpose of the present invention is to overcome the shortcomings of the prior art and provide a human malignant phyllodes tumor cell line HJP-0320 to fill the vacancy of phyllodes tumor cell lines derived from human populations at home and abroad. The human malignant phyllodes tumor cell line of the present invention is derived from primary phyllodes tumor cells extracted from Chinese population tissues, and can be used to study the mechanism of occurrence and development of breast phyllodes tumors and related drugs.

In order to achieve the above object, the technical solution adopted by the present invention includes the following aspects:

In the first aspect, the present invention provides a human malignant phyllodes tumor cell line HJP-0320, said cell line is preserved in the China Center for Type Culture Collection (CCTCC), the preservation address is: Wuhan University, Wuhan, China, and the preservation number is C2018233.

In the second aspect, the present invention provides a cell model for studying the mechanism of tumor occurrence and development, the cell model is the above-mentioned cell line.

Preferably, the tumor is a malignant tumor.

Preferably, the tumor is a phyllodes carcinoma of the breast.

In the third aspect, the present invention provides the application of the above-mentioned cell line HJP-0320 in screening antitumor drugs.

Preferably, the tumor is a phyllodes tumor of the breast.

In summary, the beneficial effects of the present invention are:

The human phyllodes tumor cell line HJP-0320 of the present invention is established from a Chinese source. The establishment time is short and the biological heredity is stable. At present, there are no human malignant phyllodes tumor cell lines on the market. Using this phyllodes tumor cell line as a research model is of great help in understanding the pathogenesis of Chinese phyllodes tumor patients.

Description of drawings

Fig. 1 is a photo of the human malignant phyllodes tumor cell line HJP-0320 in the present invention under an optical microscope.

Detailed ways

The invention relates to the field of microbial animal cell lines, in particular to a human breast phyllodes tumor cell line and its establishment method. The human phyllodes tumor cell line HJP-0320 in the present invention is derived from the right mammary tumor of a 47-year-old female patient with malignant phyllodes tumor.

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental methods in the present invention are conventional methods. Unless otherwise specified, the reagent concentrations in the present invention are mass concentrations.

Example 1

The human phyllodes tumor cell line HJP-0320 of the present invention is obtained by the following method:

(1) Specimen collection and storage methods: After the complete mammary gland phyllodes tumor has been completely removed, the tumor tissue is cut in the middle with a sterile scalpel, and the vigorously proliferating tissue is taken in DMEM medium. The collection of specimens was carried out under the guidance of the surgeon and the pathologist to prevent the diagnosis of the pathology report from being affected; when the specimens were not used for follow-up operations, they were stored in DMEM medium at 4°C, and the specimens should be used within 24 hours as much as possible.

(2) Primary culture: the tissue was washed 3 times with PBS, mechanically cut to as small a size as possible, digested with type III collagenase (1mg/ml, Worthington) and added to a 50ml centrifuge tube containing DMEM/F12, protected from light with tinfoil, 37°C, 180 rpm, digested for 1 hour, centrifuged at 250g x 5min, washed once with PBS, inoculated, subcultured and observed. Cell culture conditions: 37°C, 5% (V/V) CO₂Cell incubator, culture medium used is: DMEM/F12 (Invitrogen); EGF (Peprotech, 20ng/ml); Hydrocortisone (Sigma, 0.5mg/ml); Insulin (Sigma, 10ug/ml); Its ER(-), PR(-), Her-2(-); when subcultured, the cells were digested with Tryple trypsin, and the ratio of 1:2 was used for subculture.

(3) Purified cells: In order to extract purer primary phyllodes tumor cells derived from mesenchymal components, after the cells have been passaged for several times, the cells are subcultured to a larger culture dish at a ratio of 1:10 for culture, and the cells with mesenchymal characteristics are observed and marked under a microscope.

(4) Immortalization of primary cells: HJP-0320 cell line is infected with the immortalized virus SV40T, so that the primary cells can be successfully infected and passed down to 30 generations, that is, the immortalization is successful (this method comes from abm company).

Among them, the specific steps of immortalization are as follows:

1. Cultivate packaging cells: Before transfection, resuscitate the lentiviral packaging cells 293T in advance and culture them on a 10cm cell plate, add 10ml of DMEM medium containing 10% (W/W) heat-inactivated fetal bovine serum (FBS) to make the cells reach a 70-80% fusion rate during virus packaging. The cell culture conditions are the same as the above step (2) primary culture.

2. Prepare the lentiviral mixture:

2.1. Using LIP3000 method for transfection, add 10ug pLenti-SV40-TVector plasmid (from abm company) and 10ug lentiviral transfection packaging plasmid to 750ul opti-mem, mix well, and incubate at room temperature for 5min. At the same time, add 80ul LIP3000 transfection reagent to 750ul opti-mem, mix well, and incubate at room temperature for 5min;

2.2. Add the LIP3000 transfection reagent mixed solution into the plasmid mixed solution, mix well, and incubate at room temperature for 15 minutes.

3. Replace the prepared packaging cell 293T with 6ml of fresh medium, add the lentivirus mixture, and shake well. The cells were cultured at 37° C. for 48 h under the condition of 5% (V/V) CO ₂ .

4. Harvest lentivirus: collect the culture medium 48 hours after transfection, and filter the culture medium with a 0.45 μm filter membrane to obtain purified virus liquid. The virus particles were collected by centrifugation at 10,000×g for 4 hours to obtain concentrated lentiviruses and stored in a -80°C refrigerator.

5. Lentivirus infection:

5.1 Six hours before infection, inoculate a 6-well plate to ensure that the phyllodes tumor cells grow to a confluence density of about 30-40% during infection;

5.2 Take the virus liquid stored in the -80°C refrigerator to room temperature to thaw, and mix well;

5.3 According to the virus titer and the optimum MOI value determined by the preliminary experiment, dilute the virus stock solution with the virus dilution culture medium (10% FBS complete medium, containing double antibody), prepare 0.8mg/mL polybrene, infect the cells in a 6-well plate, add 2ml virus solution (containing 10ug/ml Polybrene) to each well, put the virus supernatant in the refrigerator, and infect the second time in the afternoon;

5.4 On the second day, remove the virus liquid in the cells, add the complete culture and incubate in the incubator. After the cells are full, carry out passage, and culture the uninfected cells and the infected cells synchronously, passage for more than 30 generations, and select the cells with normal morphology for downstream detection.

Embodiment 2 detects the expression situation of virus

Q-PCR (quantitative real-time polymerase chain reaction, referred to as quantitative PCR) was used to detect the expression level of the introduction-related immortalization gene (SV40T) in the HJP-0320 cell line. Wherein, the gene primers used are shown in Table 1 below.

Table 1 Primer base sequence

Forward (forward primer) Reverse (reverse primer) GAPDH AGAGCCTCGAGGAGAAGTTCC (SEQ ID NO. 1) ACTAGGGAGTCAAGGACGGG (SEQ ID NO. 2) SV40T GCCAGTACCGTGCCTTATCC (SEQ ID NO. 3) GAACCACTAGGCCCATAACCA (SEQ ID NO. 4)

The HJP-0320 cell line (preservation number C2018233) was introduced with SV40T virus, and the overexpression detection results of SV40T in the HJP-0320 cell line are shown in Table 2 below.

Table 2 Expression results of SV40T in HJP-0320 cell line

sample Introduced gene ct value whether to express control group GAPDH 14 yes control group SV40T 35 no immortalization group GAPDH 14 yes immortalization group SV40T 21.22 yes

From the data in Table 2 above, it can be seen that the SV40T gene (introduction method refers to the virus infection step in Example 1) in the control group cells (wild-type breast phyllodes tumor cells) cannot be expressed, while the inserted gene SV40T in the immortalized group cells (HJP-0320) has been successfully expressed, indicating that the HJP-0320 cells of the present invention have been successfully immortalized.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limiting the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims (2)

1. A human malignant foliar tumor cell line HJP-0320, wherein the cell line is deposited with the chinese collection at the following address: china, university of Wuhan, and preservation number is CCTCC NO: C2018233.

2. a cell model for researching a tumorigenic development mechanism, which is a human malignant foliate tumor cell line HJP-0320; the tumor is a lobate malignant tumor of the breast.