CN111019897A

CN111019897A - Human benign leaf-shaped tumor cell line GLK-1010 and application thereof

Info

Publication number: CN111019897A
Application number: CN201910115537.5A
Authority: CN
Inventors: 宋尔卫; 聂燕; 黄红颜
Original assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Current assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Priority date: 2019-02-14
Filing date: 2019-02-14
Publication date: 2020-04-17
Anticipated expiration: 2039-02-14
Also published as: CN111019897B

Abstract

The invention discloses a human benign leaf tumor cell line GLK-1010, which is preserved in China center for type culture Collection with the preservation addresses as follows: china, Wuhan university, with the accession number C2018235. The invention also discloses a cell model of human benign leaf tumor cell line GLK-1010 as a research tumor occurrence and development mechanism and application in screening antitumor drugs. The human phylliform tumor cell line GLK-1010 is established from Chinese, the establishment time is short, the biological heredity is stable, and the benign phylliform tumor cell line of human is lacked in the market at present.

Description

Human benign leaf-shaped tumor cell line GLK-1010 and application thereof

Technical Field

The invention relates to the technical field of tumor cells, in particular to a human benign phylliform tumor cell line GLK-1010 and application thereof.

Background

The breast phyllodes tumor is a rare breast tumor, which accounts for about 1 percent of the breast tumor, and the tumor grows quickly and is often expressed as a huge tumor; histologically, it is classified as benign, junctional, malignant; even benign leaf tumors recur, and malignant leaf tumors also undergo hematogenous metastasis. Chemotherapy and radiotherapy have uncertain therapeutic effects on leaf tumors, and the existing treatment method capable of reducing the recurrence and metastasis probability is enlarged surgical resection, but even if enlarged surgical resection is carried out, the local recurrence rate of the leaf tumors is still as high as 8-36%, and the blood-borne metastasis rate of malignant leaf tumors is as high as 22%.

At present, the mechanism that contributes to the malignant transformation of phyllodes is not clear. Existing molecular markers are also of limited value in predicting the biological behavior of tumors. Therefore, it is necessary to enhance the research on the malignant progression mechanism of the leaf tumor cells, and the method has important significance in suppressing the malignant progression of the leaf tumor and reducing the local recurrence, distant metastasis and mortality of the malignant leaf tumor. The establishment of reliable leaf-shaped tumor cell lines is urgent.

Disclosure of Invention

Based on the problems, the invention aims to overcome the defects of the prior art and provide a human benign phylliform tumor cell line GLK-1010 to fill the vacancy of the phylliform tumor cell line derived from the current domestic and foreign populations.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following aspects:

in a first aspect, the invention provides a human benign leaf tumor cell line GLK-1010, which is deposited at the chinese type culture collection with the following addresses: china, Wuhan university, with the accession number C2018235.

In a second aspect, the present invention provides a cell model for studying the mechanism of tumorigenesis and development, said cell model being a cell line as described above.

Preferably, the tumor is a benign tumor.

Preferably, the tumor is a benign leaf tumor of the breast.

In a third aspect, the invention provides an application of the cell line GLK-1010 in screening antitumor drugs.

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

In conclusion, the beneficial effects of the invention are as follows:

the human phylliform tumor cell line GLK-1010 is established from Chinese, the establishment time is short, the biological heredity is stable, and the benign phylliform tumor cell line of human is lacked in the market at present.

Drawings

FIG. 1 is a photograph of human benign leaf tumor cell line GLK-1010 under an optical microscope.

Detailed Description

The invention relates to the field of microbial animal cell lines, in particular to a human breast phylliform tumor cell line and an establishment method thereof. The human phylliform tumor cell line GLK-1010 is derived from the right breast tumor of a 46-year-old female patient with benign phylliform tumor, is named as human phylliform tumor benign cell line GLK-1010, is preserved in a Chinese typical culture center, and has the preservation date of 2018, 12 months and 16 days; the preservation address is as follows: china, Wuhan university, with a preservation number of CCTCC NO: C2018235.

to better illustrate the objects, aspects 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 specified, the concentrations of the reagents in the present invention are mass concentrations.

Example 1

The human phylliform tumor cell line GLK-1010 is obtained by the following method:

(1) the specimen collecting and storing method comprises the following steps: when the complete breast phylliform tumor is completely removed by operation, a sterile scalpel is used for cutting the center of the tumor tissue, and the tissue with vigorous and active hyperplasia is taken in a DMEM culture medium. The collection of the specimens is carried out under the guidance of a main scalpel and a pathologist, so that the influence on the diagnosis of pathological reports is prevented; when the specimen was not used for the subsequent operation, it was stored in DMEM medium at 4 ℃ and used for as long as 24 hours.

(2) Primary culture: the tissue was washed 3 times with PBS, mechanically sheared to the greatest extent, collagenase iii digestion (1mg/ml, Worthington) was added to a 50ml centrifuge tube containing DMEM/F12, tinfoil paper was protected from light, 37 degrees, 180 rpm, digested for 1 hour, centrifuged for 250g x 5min, PBS washed 1 time, inoculated and subcultured and observed cultured under the cell culture conditions: 37 ℃ and 5% (V/V) CO₂The cell culture box adopts the following culture media: DMEM/F12 (Invitrogen); EGF (Peprotech,20 ng/ml); hydrocortisone (Sigma,0.5 mg/ml); insulin (Sigma,10 ug/ml); pen/strep (Invitrogen); the cell morphology of cell line GLK-1010 during culture is shown in FIG. 1: long spindle, fibroepithelial tumors; when the cells are passaged, the cells are digested by Tryple trypsin and are passaged according to the ratio of 1: 2.

(3) And (3) purifying cells: in order to extract primary phyllo-tumor cells from relatively pure interstitial components, specifically, after the cells are passaged for several times, the cells are passaged to a large culture dish according to the ratio of 1:10 to be cultured, cells with interstitial characteristics are observed and marked under a mirror, cells with peripheral epithelial characteristics are scraped under a sterile condition, washed by PBS for 3 times, and added with a fresh culture medium to be continuously cultured and passaged.

(4) Immortalizing primary cells: GLK-1010 cell line successfully infected primary cells and passed to 30 passages by infecting the immortalizing virus SV40T, i.e., immortalization was successful (this method was from abm).

Wherein the immortalization comprises the following specific steps:

1. culturing the packaging cells: before transfection, the lentivirus packaging cell 293T is recovered in advance and cultured in a 10cm cell plate, and 10ml of DMEM medium containing 10% (W/W) heat-inactivated Fetal Bovine Serum (FBS) is added to ensure that the cell fusion rate reaches 70-80% when the virus packaging is carried out. The cell culture conditions were the same as in the primary culture of step (2).

2. Preparing a lentivirus mixture:

2.1. transfection was carried out using the method of LIP3000, adding 10ug of pLenti-HPV Vector plasmid (from abm) and 10ug of lentiviral transfection packaging plasmid to 750ul of opti-mem, mixing well, and incubating at room temperature for 5 min. Simultaneously adding 80ul of LIP3000 transfection reagent into 750ul of opti-mem, mixing evenly, and incubating for 5min at room temperature;

2.2. adding the LIP3000 transfection reagent mixed solution into the plasmid mixed solution, mixing uniformly, and incubating at room temperature for 15 min.

3. The prepared packaging cells 293T were replaced with 6ml of fresh medium, the lentivirus mixture was added and shaken well. Cells were at 5% (V/V) CO₂Culturing at 37 deg.C for 48 h.

4. And (3) harvesting lentivirus: the medium was collected 48 hours after transfection, and the medium was filtered through a 0.45 μm filter to obtain a purified virus solution. The virus particles were collected by centrifugation at 10,000 Xg for 4 hours to obtain concentrated lentiviruses, which were stored in a freezer at-80 ℃.

5. Lentivirus infection:

5.1 inoculating 6-hole plate 6h before infection to ensure that the leaf-shaped tumor cells grow to about 30-40% of fusion density during infection;

5.2 taking the virus liquid stored in a refrigerator at minus 80 ℃ to room temperature for melting and mixing evenly;

5.3 according to the virus titer and the best MOI value determined by preliminary experiments, diluting the virus stock solution with virus dilution culture solution (10% FBS complete culture medium containing double antibody), preparing 0.8mg/mL Polybrene, infecting cells in a 6-well plate, adding 2mL of virus solution (containing 10ug/mL Polybrene) into each well, placing the virus supernatant in a refrigerator, and secondarily infecting in the afternoon;

and 5.4, removing virus liquid in the cells, adding the virus liquid into a complete culture medium incubator for incubation, carrying out passage after the cells grow full, synchronously culturing the uninfected cells and the infected cells for more than 30 passages, and selecting the cells with normal shapes for downstream detection.

Example 2 detection of viral expression

The expression level of the immortalized gene (HPV) introduced into the cell line GLK-1010 is detected by Q-PCR (quantitative real-time polymerase chain reaction, quantitative PCR for short), wherein the primer sequences for amplifying the corresponding genes are shown in the following table 1.

TABLE 1 primer base sequences

	Forward (Forward primer)	Reverse (Reverse primer)
			GAPDH	AGAGCCTCGAGGAGAAGTTCC(SEQ ID NO.1)	ACTAGGGAGTCAAGGACGGG(SEQ ID NO.2)
HPV	TAACTTCTGTTCGGGGAGGC(SEQ ID NO.3)	AGGGAGGGAAAAGATCCCGA(SEQ ID NO.4)

The GLK-1010 cell line (accession number C2018235) introduced HPV virus, and the results of detection of HPV overexpression in the GLK-1010 cell line are shown in Table 2 below.

TABLE 2 HPV expression in GLK-1010 cells

Sample (I)	Introduction of Gene	ct value	Whether or not to express
				Control group	GAPDH	14	Is that
Control group	HPV	34	Whether or not
				Immortalizing group	GAPDH	13	Is that
Immortalizing group	HPV	21.93	Is that

As is clear from the results shown in Table 2 above, HPV genes (introduction method in reference to the virus infection step in example 1) were not expressed in the cells of the control group (wild-type breast phylliform tumor cells), while HPV genes inserted in the immortalized breast phylliform tumor cells (GLK-1010 cells) were successfully expressed, indicating the successful immortalization of GLK-1010 cells according to the present invention.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is 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 on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Human benign leaf tumor cell line GLK-1010, wherein said cell line is deposited at the China center for type culture Collection with the following addresses: china, Wuhan university, with the accession number C2018235.

2. A cell model for studying the mechanism of development of tumorigenesis, said cell model being the cell line of claim 1.

3. The cell model of claim 2, wherein the tumor is a benign tumor.

4. The cell model of claim 2 or 3, wherein the tumor is a benign leaf tumor of the breast.

5. The use of the cell line GLK-1010 of claim 1 in screening for anti-tumor drugs.

6. The use according to claim 5, wherein the tumor is a benign tumor of the breast.