CN111019898B - Human malignant foliar tumor cell line HJP-0320 and application thereof - Google Patents

Human malignant foliar tumor cell line HJP-0320 and application thereof Download PDF

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CN111019898B
CN111019898B CN201910115538.XA CN201910115538A CN111019898B CN 111019898 B CN111019898 B CN 111019898B CN 201910115538 A CN201910115538 A CN 201910115538A CN 111019898 B CN111019898 B CN 111019898B
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CN111019898A (en
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聂燕
宋尔卫
黄红颜
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Sun Yat Sen Memorial Hospital Sun Yat Sen University
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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 foliar tumor cell line HJP-0320 and application thereof
Technical Field
The invention relates to the technical field of tumor cell lines, in particular to a human malignant foliate tumor cell line HJP-0320 and application thereof.
Background
The lobed tumor of the breast is a rare breast tumor, accounting for about 1 percent of the breast tumor, and the tumor grows quickly and is often expressed as a huge tumor; histologically classifying it into benign, borderline and malignant; even benign phylliform tumors recur, and malignant phylliform tumors undergo metastasis. The treatment effect of chemotherapy and radiotherapy on the foliar tumor is not exact, the current treatment method capable of reducing the recurrence rate and the metastasis rate of the foliar tumor is expanded surgical excision, but even if the treatment method is expanded surgical excision, the local recurrence rate of the foliar tumor is still 8-36%, and the blood-borne metastasis rate of malignant foliar tumor is up to 22%.
At present, the mechanism for promoting malignant transformation of leaf tumors is not clear. The value of existing molecular markers in predicting the biological behaviour of tumors is also limited. Therefore, the enhancement of research on malignant progress mechanisms of the leaf tumor cells is very necessary, and has important significance for suppressing malignant progress of the leaf tumor, reducing local recurrence, distant metastasis and mortality of the malignant leaf tumor. It is urgent to establish reliable leaf tumor cell lines.
Disclosure of Invention
Based on the above problems, the present invention aims to overcome the defects of the prior art and provide a human malignant foliar tumor cell line HJP-0320 to fill the current gap of foliar tumor cell lines from domestic and foreign people, and the human malignant foliar tumor cell line of the present invention is derived from primary foliar tumor cells extracted from Chinese crowd tissues, and can be used for researching mammary gland foliar tumor generation and development mechanisms and related drugs.
In order to achieve the above purpose, the technical scheme adopted by the invention comprises the following aspects:
in a first aspect, the invention provides a human malignant foliar tumor cell line HJP-0320, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation address: china, university of Wuhan, and preservation number is C2018233.
In a second aspect, the invention provides a cell model for studying the mechanism of tumorigenesis, said cell model being the cell line described above.
Preferably, the tumor is a malignant tumor.
Preferably, the tumor is a breast phylliform malignancy.
In a third aspect, the invention provides the application of the cell line HJP-0320 in screening anti-tumor drugs.
Preferably, the tumor is a breast phylliform tumor.
In summary, the beneficial effects of the invention are as follows:
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 tumor patients by taking the foliar tumor cell line as a research model.
Drawings
FIG. 1 is a photograph of a human malignant foliar tumor cell line HJP-0320 of the present invention under an optical microscope.
Detailed Description
The invention relates to the field of microbial animal cell lines, in particular to a human mammary gland phylliform tumor cell line and an establishment method thereof. The human foliar tumor cell line HJP-0320 in the invention is derived from a right breast tumor of a 47-year-old female patient suffering from malignant foliar tumor, is named as a human foliar tumor malignant cell line HJP-0320, and is preserved in China center for type culture Collection with a preservation date of 2018, 12 months and 16 days; the preservation address is: china, university of Wuhan, and preservation number is CCTCC NO: C2018233.
for a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental methods in the present invention are all conventional methods. Unless otherwise indicated, the concentrations of the reagents in the present invention are mass concentrations.
Example 1
The human phylliform tumor cell line HJP-0320 is obtained by the following method:
(1) The sample collection and preservation method comprises the following steps: and after the complete mammary gland phylliform tumor is removed by a complete operation, a sterile scalpel is used for centrally cutting the tumor tissue, and the tissue with vigorous activity and active hyperplasia is taken out in a DMEM culture medium. The collection of the specimens is carried out under the guidance of a surgical main knife and a pathologist, so that the influence on the diagnosis of a pathological report is prevented; when the specimen is not subjected to subsequent operation, the specimen is stored in a DMEM medium at the temperature of 4 ℃ and used for 24 hours as much as possible.
(2) Primary culture: the tissue was washed 3 times with PBS, mechanically sheared to the extent possible, type III collagenase digested (1 mg/ml, worthington) added to a 50ml centrifuge tube containing DMEM/F12, tinfoil protected from light, 37 degrees, 180 rpm, digested for 1 hour, centrifuged 250g x 5min, washed 1 time with PBS, inoculated and subcultured and observed for cell culture conditions: 37 ℃,5% (V/V) CO 2 The cell incubator is used for culturing the following culture mediums: DMEM/F12 (Invitrogen); EGF (Peprotech, 20 ng/ml); hydrocritisone (Sigma, 0.5 mg/ml); insulin (Sigma, 10 ug/ml); pen/Strep (Invitrogen); the cell morphology of the cell line HJP-0320 during the culture is shown in FIG. 1: long fusiform, high invasive mesenchymal cells grow faster, ER (-), PR (-), her-2 (-); at passage, cells were digested with Tryple pancreatin and passaged at a ratio of 1:2.
(3) Purifying the cells: in order to extract primary leaf tumor cells from purer interstitial components, specifically, after the cells are passaged for several times, the cells are passaged into a larger culture dish according to the ratio of 1:10 for culture, the cells with interstitial characteristics are observed under a lens and marked, the cells with peripheral epithelial characteristics are scraped under the aseptic condition, washed for 3 times by PBS, and fresh culture medium is added for continuous culture and passaging.
(4) Immortalization of primary cells: the HJP-0320 cell line was successfully infected with the immortalized virus SV40T and was passaged to 30 passages, i.e.immortalized, by infection (this method was available from abm company).
Wherein, the immortalization specifically comprises the following steps:
1. culturing packaging cells: prior to transfection, lentiviral packaging cells 293T were resuscitated in 10cm plates and incubated, 10ml of DMEM medium containing 10% (W/W) heat-inactivated Fetal Bovine Serum (FBS) was added to achieve a fusion rate of 70-80% of the cells when they were virus packaged. The cell culture conditions are the same as those of the primary culture in the step (2).
2. Preparing a lentiviral mixture:
2.1. transfection was performed by LIP3000, and 10ug of pLenti-SV40-T Vector plasmid (from abm Co.) and 10ug of lentiviral transfection packaging plasmid were added to 750ul opti-mem, mixed, and incubated at room temperature for 5min. Meanwhile, 80ul of LIP3000 transfection reagent is added into 750ul opti-mem, and the mixture is uniformly mixed and incubated for 5min at room temperature;
2.2. adding the LIP3000 transfection reagent mixed solution into the plasmid mixed solution, mixing uniformly, and incubating for 15min at room temperature.
3. The prepared packaging cell 293T was replaced with 6ml of fresh medium, and the lentiviral mixture was added and shaken well. Cells at 5% (V/V) CO 2 Culturing at 37℃for 48h.
4. Harvesting lentiviruses: the culture medium was collected 48 hours after transfection, and was filtered through a 0.45 μm filter membrane to obtain a purified virus solution. The virus particles were collected by centrifugation at 10,000Xg for 4 hours to obtain concentrated lentiviruses, which were stored in a-80℃refrigerator.
5. Lentiviral infection:
5.1 6 hours before infection, inoculating a 6-hole plate, and ensuring that leaf tumor cells grow to about 30-40% of fusion density during infection;
5.2, taking the virus liquid stored in a refrigerator at the temperature of minus 80 ℃ to the room temperature for melting, and uniformly mixing;
5.3 diluting the virus stock with a virus dilution medium (10% FBS complete medium, containing double antibodies) according to the virus titer and the optimal MOI value determined by the preliminary experiment, preparing 0.8mg/mL Polybrene, infecting cells in 6-well plates, adding 2mL of virus solution (containing 10ug/mL Polybrene) per well, placing the virus supernatant in a refrigerator, and carrying out secondary infection in the afternoon;
5.4, removing virus liquid in cells the next day, adding the complete culture, incubating in an incubator, carrying out passage after the cells grow up, synchronously culturing uninfected cells and infected cells, carrying out passage for more than 30 generations, and selecting cells with normal morphology for downstream detection.
Example 2 detection of viral expression
The expression level of the introduced related immortalized gene (SV 40T) of the HJP-0320 cell line was detected by Q-PCR (quantitative real-time polymerase chain reaction, abbreviated as quantitative PCR). Among them, the gene primers used are shown in Table 1 below.
TABLE 1 primer base sequences
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 SV40T virus was introduced into the HJP-0320 cell line (accession No. C2018233), and the results of the detection of overexpression of SV40T in the HJP-0320 cell line are shown in Table 2 below.
TABLE 2 results of SV40T expression in HJP-0320 cell lines
Sample of Introduction of genes ct value Whether or not to express
Control group GAPDH 14 Is that
Control group SV40T 35 Whether or not
Immortalized group GAPDH 14 Is that
Immortalized group SV40T 21.22 Is that
As is clear from the data in Table 2, SV40T gene (the virus infection step in reference example 1 for the introduction method) was not expressed in the control cells (wild-type mammary gland foliate tumor cells), but SV40T gene inserted in the immortalized cells (HJP-0320) was successfully expressed, which indicates that the immortalization of HJP-0320 cells of the present invention was successful.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution 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.
CN201910115538.XA 2019-02-14 2019-02-14 Human malignant foliar tumor cell line HJP-0320 and application thereof Active CN111019898B (en)

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CN113293133B (en) * 2021-03-05 2023-06-20 中山大学孙逸仙纪念医院 Human breast malignant phylliform tumor cell strain and application thereof
CN113801849B (en) * 2021-07-14 2023-11-07 中山大学孙逸仙纪念医院 Human breast benign phylliform tumor cell strain BPT-0526 and application thereof
CN114717190B (en) * 2022-04-20 2023-10-03 中山大学孙逸仙纪念医院 Human breast malignant phylliform tumor cell line BPT0713 and application thereof
CN116121191A (en) * 2022-09-08 2023-05-16 中山大学孙逸仙纪念医院 Human breast malignant phylliform tumor cell line SYSH-MPT-04 and application thereof

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