阅读说明：本技术 一种犬乳腺癌细胞系、其建立方法及应用 (Canine breast cancer cell line, and establishment method and application thereof ) 是由 梅晨 王宏俊 张雪 先宏 于 2021-08-19 设计创作，主要内容包括：本发明提供了一种犬乳腺癌细胞系、其建立及应用。该犬乳腺癌细胞系犬乳F17,保藏号为CGMCC No.22349,保藏日期为2021年05月26日；保藏单位名称为中国微生物菌种保藏管理委员会普通微生物中心。本发明提供的细胞系细胞呈多极形上皮形态特征,聚集成片状或团簇状,贴壁牢固,难消化分离。细胞接种成瘤率和自发性肺转移为100％,适用于犬乳腺癌的体内体外试验研究,为肿瘤发生发展机制研究、药物靶点研究、为药物治疗机制研究提供很好体外试验模型,具有重要的基础研究与临床应用价值。(The invention provides a canine breast cancer cell line, and establishment and application thereof. The dog breast cancer cell line dog milk F17 has the preservation number of CGMCC No.22349 and the preservation date of 2021, 05 and 26 days; the name of the preservation unit is China general microbiological culture Collection center. The cell line cells provided by the invention are characterized by a multipole epithelial morphology, are aggregated into a sheet shape or a cluster shape, are firmly attached to the wall, and are difficult to digest and separate. The cell inoculation tumor forming rate and spontaneous lung metastasis are 100 percent, the kit is suitable for in-vivo and in-vitro experimental research of canine breast cancer, provides a good in-vitro experimental model for tumor occurrence development mechanism research, drug target point research and drug treatment mechanism research, and has important basic research and clinical application values.)

1. The canine breast cancer cell line is characterized in that the canine breast cancer cell line is canine milk F17 with the preservation number of CGMCC No.22349 and the preservation date of 2021, 05 and 26 days; the name of the preservation unit is China general microbiological culture Collection center, and the address of the preservation unit is No. 3 Xilu No. 1 Beijing.

2. The canine breast cancer cell line of claim 1, wherein the canine breast cancer cell line is capable of long-term growth and stable passage in vitro, and has biological and genetic characteristics of: (1) the cells are in the shape of a multipole epithelium, are aggregated into a sheet or a cluster, are firmly attached to the wall, and are difficult to digest, separate and contact with growth inhibition loss; (2) the cells are of canine origin, and the number of chromosomes is 78; (3) the cell inoculation tumor formation rate and the spontaneous lung metastasis rate are 100 percent, and the clinical characteristics of the canine breast cancer can be reproduced.

3. The canine breast cancer cell line of claim 2, wherein the clinical profile is log-proliferative phase for 2-4 days, positive for immunofluorescence Ki-67, SOX-2, DELTA-CATENIN, CLAUDIN-1; after cell cryopreservation and recovery, the cells grow well and can be continuously passaged.

4. A preparation method of a canine breast cancer cell line comprises the following steps:

(1) case tumor isolation: aseptically cutting breast tumor tissues of clinical dogs;

(2) primary culture of tumor cells:

tumor tissue was washed 3 times by trituration with PBS containing 1% penicillin streptomycin solution, and then, the tumor was treatedCollecting debris, decomposing with collagenase II, centrifuging, collecting cells, suspending in DMEM/F12 complete medium containing 10% fetal calf serum, adding 1% streptomycin, and suspending the cells in 5% CO₂Culturing in a cell culture box;

(3) establishing a cell line:

when the cell culture reaches 90% fusion, washing with PBS for 3 times, then digesting with 0.25% trypsin, extracting secondary culture from the digested cell, changing into new culture solution to continue culturing, carrying out passage for 1 time every 3d with the seed separation rate of 1:3, carrying out in vitro passage to 50 generations, and detecting and verifying biological indexes.

5. The method of claim 4, wherein the biological marker assay comprises cellular morphology, chromosomal analysis, tumor formation rate, and growth pattern.

6. The preparation method of claim 5, wherein the cell morphology is that the cancer cells are characterized by a multipolar epithelial morphology, are aggregated into a sheet or cluster shape, are firmly attached to the wall of a culture flask and are difficult to digest and separate, the chromosome analysis shows that the chromosome number is 78, the tumor formation rate is 100%, and the tumor growth conforms to a Gompertzian index function model.

7. Use of a canine breast cancer cell line of any one of claims 1-3 in the preparation of a model for generating cancer in an immunodeficient mammal.

8. The use of claim 7, wherein the cancer model is a nude mouse tumor-bearing model.

9. The use of claim 8, wherein the canine breast cancer cell line is subcutaneously inoculated in nude mice in an amount of 10⁸And (4) cells.

Technical Field

The invention belongs to the technical field of microbial animal cell lines, and particularly relates to a canine breast cancer cell line, and establishment and application thereof.

Background

Triple Negative Breast Cancer (TNBC) accounts for approximately 15% of all breast cancer cases and does not express the Progestogen Receptor (PR), the Estrogen Receptor (ER) and the human epidermal growth factor receptor 2(HER 2). Thus, hormone therapy is not beneficial to them. The overall survival rate for TNBC patients was lower than for non-TNBC patients.

Canine breast Cancer (CMT) is one of the most common tumors in uninfected bitches. In the clinic, CMT is the second largest tumor to skin tumors only. Since CMT has similar epidemiological, histological, and clinical features as HBC, about 50% of CMT cases are malignant. CMT has been used as a good model for studying human breast cancer.

At present, the molecular mechanism of the canine breast cancer pathogenesis is not very clear, and no ideal treatment scheme or medicament exists. Cell lines can be used in many mechanistic studies, which provide a good platform for studying cell invasion and metastasis mechanisms. Because the cell sources of the dogs are different, the canceration genes are also different, and each cell line is unique in existence and has specificity in cancer research. The established canine breast cancer cell lines are quite limited at present, and the canine triple negative breast cancer cell lines have not been reported. In addition, the defects of instability, low inoculation tumor rate and the like of the prepared canine breast cancer cell line in the prior art exist, so that the development of a CMT cell line which has stability and can reproduce the clinical characteristics of canine triple negative breast cancer is urgently needed.

Disclosure of Invention

In order to solve the technical problems, the invention provides a canine breast cancer cell line and a tumor-bearing mouse successfully, and provides a cell model and a research tool for researching the pathogenesis of canine breast cancer and the tumor metastasis molecular mechanism. The invention also provides an establishment method of the canine breast cancer cell line capable of stably forming tumors in nude mice.

In order to achieve the purpose, the invention adopts the following technical scheme that:

the canine breast cancer cell line is named as canine milk F17, the preservation number is CGMCC No.22349, and the preservation date is 2021, 05 and 26 days; the name of the preservation unit is China general microbiological culture Collection center (CGMCC), the preservation unit is CGMCC for short, and the address of the preservation unit is No. 3 of Xilu No. 1 of Beijing, Chaoyang.

Preferably, the canine breast cancer cell line, capable of long-term growth and stable passage in vitro, has the following biological and genetic characteristics: (1) the cells are in the shape of a multipole epithelium, are aggregated into a sheet or a cluster, are firmly attached to the wall, and are difficult to digest, separate and contact with growth inhibition loss; (2) the cells are of canine origin, and the number of chromosomes is 78; (3) the cell inoculation tumor formation rate and the spontaneous lung metastasis rate are 100 percent, and the clinical characteristics of the canine breast cancer can be reproduced.

Preferably, the clinical characteristics are that the logarithmic proliferation phase is 2-4 days, and the immunofluorescence Ki-67, SOX-2, DELTA-CATENIN, CLAUDIN-1 are positive; after cell cryopreservation and recovery, the cells grow well and can be continuously passaged.

The preparation method of the canine breast cancer cell line comprises the following steps:

(1) case tumor isolation: aseptically cutting breast tumor tissues of clinical dogs;

(2) primary culture of tumor cells:

grinding and washing tumor tissue with PBS containing 1% penicillin streptomycin solution for 3 times, collecting tumor debris, decomposing with collagenase II, centrifuging, collecting cells, suspending in DMEM/F12 complete medium containing 10% fetal calf serum, adding 1% penicillin streptomycin, and suspending in 5% CO₂Culturing in a cell culture box;

(3) establishing a cell line:

when the cell culture reaches 90% fusion, washing with PBS for 3 times, then digesting with 0.25% trypsin, extracting secondary culture from the digested cell, changing into new culture solution to continue culturing, carrying out passage for 1 time every 3d with the seed separation rate of 1:3, carrying out in vitro passage to 50 generations, and detecting and verifying biological indexes.

In the preparation method described above, preferably, the biological indicator measurement includes cell morphology, chromosome analysis, tumor formation rate and growth pattern. After the cells are purified, the cells can stably grow, the cell morphology is uniform, the cell fluorescent staining observation shows that the cancer cell marker is positively expressed and accords with the characteristics of the cancer cells, and the inoculated cells can grow in a mouse body to make the mouse tumor.

In the preparation method, preferably, the cell morphology is that the cancer cells are characterized by a multipolar epithelial morphology, are aggregated into sheets or clusters, are firmly attached to the wall of a culture flask and are difficult to digest and separate, the chromosome number is 78 through chromosome analysis, the tumor formation rate is 100%, and the tumor growth conforms to a Gompertzian index function model.

Use of a canine breast cancer cell line as described above for the preparation of a model for generating cancer in an immunodeficient mammal with a neoplasia rate of 100%.

The use as described above, the immunodeficient mammal producing the cancer model is a nude mouse tumor-bearing model.

As described above, the canine breast cancer cell line was subcutaneously inoculated in nude mice in an amount of 10⁸And (4) cells.

The progeny cells of the canine breast cancer cell line, substantially or completely retained the characteristics of the parent cell.

The invention has the beneficial effects that:

the canine breast cancer cell line provided by the invention can be used for successfully establishing a nude mouse tumor-bearing model.

The invention utilizes clinical canine primary breast cancer to establish a tumor source, performs cell separation and purification culture, adopts an in vitro culture mode to passage, establishes a canine breast cancer cell line, and can successfully establish a nude mouse tumor bearing model by utilizing the cell line. The cell line cells of the invention are characterized by a multi-polar epithelial morphology, are aggregated into a sheet shape or a cluster shape, are firmly attached to the wall, and are difficult to digest and separate. The cell inoculation tumor forming rate and spontaneous lung metastasis are 100 percent, the clinical characteristics of the triple negative breast cancer are reproduced, the stability is high, the model is an ideal and reliable research model, the model is suitable for in vivo and in vitro experimental research of the canine triple negative breast cancer, a good in vitro experimental model is provided for tumor occurrence development mechanism research, drug target point research and drug treatment mechanism research, and the model has important basic research and clinical application values.

Drawings

FIG. 1 shows the cell morphology of canine milk F17 under an inverted microscope.

FIG. 2 shows chromosome of canine milk F17 cell line.

FIG. 3 shows a tumor-bearing model of nude mice inoculated subcutaneously with canine milk F17 cells.

FIG. 4 shows tumor-bearing histopathological morphology (HE X400) of nude mice inoculated subcutaneously with canine milk F17 cells.

FIG. 5 shows the results of immunofluorescence (Ki-67, SOX-2, DELTA-CATENIN, CLAUDIN-1) measurements of canine milk F17 cells.

Detailed Description

The following examples are intended to further illustrate the invention but should not be construed as limiting it. Modifications and substitutions may be made thereto without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and unless otherwise specified, the reagents used in the present invention are analytically pure or above.

Example 1 establishment of canine breast cancer cell lines

1. Isolation of primary tumors

By surgical resection, a mammary tumor was obtained from a 5-year-old addison dog, clinically and histopathologically diagnosed as Canine Mammary Tumor (CMT): with firm, thickened and painful clinical manifestations. After surgical extraction, tumor specimens were rapidly soaked with Phosphate Buffered Saline (PBS) and penicillin streptomycin solution (Gibco, usa).

2. Primary culture of tumor cells

The tumor tissue obtained above was washed 3 times by trituration with PBS containing 1% penicillin streptomycin solution (Gibco, USA). Then, the tumor fragments were collected in a 15 ml centrifuge tube, and 0.2% collagenase II (Sigma, V900892) was added to a tissue volume of 3-4 times at 37 ℃ for 2 hours in a shaker containing 5% carbon dioxide, centrifuged at room temperature for 5 minutes at 1000rpm, the cells were collected, resuspended in 10% fetal bovine serum in DMEM/F12 complete medium (Gibco, USA), 1% penicillin, the cell suspension was added to a 25cm square flask (Corning, USA), and 5% CO was added at 37 ℃ to obtain a cell suspension₂Cultured in a cell culture box. Cells were observed daily with a microscope.

3. Establishing a cell line:

when the cell culture reached 90% confluence, the cells were washed 3 times with PBS and then treated with 2ml of complete medium containing 0.25% trypsin (HyClone, USA) and 0.02% EDTA. Extracting secondary culture from digested cells, and replacing with new 25cm²In bottle, concentration is 1x10⁵cells/ml, complete medium resuspended in DMEM/F12 containing 15% fetal bovine serum. Continuously culturing, carrying out passage 1 time every 3d, and the seed separation rate is 1: 3. The epithelial cells can be purified by utilizing the characteristic that the adherence speed of the fibroblasts is obviously higher than that of the epithelial cells. The cells primarily purified by differential adherence method are normally digested to prepare single cell suspension. The cell concentration was calculated on a cell counting plate. The cell concentration was adjusted to 10 cells per 100. mu.L with complete medium, mixed well and added well to a 96-well plate at 100. mu.L/well. And replacing the fresh culture medium every 3d, and culturing for 10-14d to show that cell colonies can grow out of a plurality of holes, namely the purified canine breast cancer cell line which is marked as canine milk F17.

Experimental observation and verification are carried out, and the purified cells shown in figure 1 are obtained by multiple times of digestion passage purification. By karyotyping, the chromosomes of the cell nucleus were extracted and observed under a microscope as shown in FIG. 2. The separated and purified cells are inoculated subcutaneously into nude mice, and all the inoculated mice form tumors, and the tumor formation rate is 100 percent, as shown in figure 3. And (3) staining the tumor-bearing mouse tumor by HE (human immunodeficiency virus) section, and observing the conditions of the breast pad tumor and the lung metastasis section, wherein A is a spontaneous lung metastasis tumor HE section of a nude mouse, and B is a breast tumor-bearing HE section of the nude mouse, as shown in figure 4. The results show that the mammary tumor model can be successfully replicated and spontaneous lung metastasis can occur when the canine F17 cells are inoculated subcutaneously into nude mice, and the success rate is 100%. The purified cells were subjected to cellular immunofluorescence assay, and the expression of tumor marker proteins such as Ki-67, SOX-2, DELTA-CATENIN, CLAUDIN-1, etc. was observed, as shown in FIG. 5. Specifically, the method comprises the following steps: cell morphology: the cancer cells are characterized by a multi-polar epithelial form, are aggregated into a sheet or cluster, are firmly attached to the wall of the culture bottle, and are difficult to digest and separate.

Chromosome analysis: 100 metaphase cells with well dispersed chromosomes were selected for chromosome analysis. After the cells grow to 90%, digesting the cells conventionally, centrifuging and collecting the cells, adding 8mL of 0.075mol/L KCl solution preheated at 37 ℃, lightly blowing and beating the cell clusters by using a suction tube, uniformly mixing, placing the cell clusters in a preheated water bath box at 37 ℃ for hypotonic treatment for 25min, adding 1mL of newly configured fixative (methanol: glacial acetic acid ═ 3:1), carefully blowing and beating by using the suction tube, uniformly mixing, centrifuging at 1000r/min for 8min, removing the supernatant, adding 8mL of fixative, blowing and beating the cell clusters to prepare a cell suspension, and then fixing at room temperature for 20 min. Centrifuging at 1000r/min for 8min, discarding the supernatant, and repeating the fixation once. Discarding the supernatant, adding several drops of newly prepared fixative according to the number of cells, and blowing to make the cells into suspension. Sucking a small amount of cell suspension, dropping 2-3 drops on a glass slide soaked by ice water, blowing away and air-drying. And (3) placing the specimen in Giemsa dye liquor, dyeing for 8min, washing with water to remove loose color, and air-drying. The results show that the chromosome number is 78, which is consistent with the chromosome number of dog cells, and the isolated cells are derived from dogs.

Tumor formation rate and growth pattern: the tumor formation rate is 100%, and the tumor growth conforms to a Gompertzian exponential function model. When the tumor of the mouse grows to 2cm³After peace, mice are examined by means of autopsy, the tumor metastasis condition is examined, multiple metastasis foci can be seen in the lung of the mice, and the spontaneous lung metastasis rate is 100%. The exponential proliferation phase of the canine milk F17 cell line was on days 2-4.

Biological characteristics of the canine milk F17 cell line: dividing the cultured cells into 2 groups, each group comprises 3 cell slide, discarding the culture solution, washing with PBS once, fixing 4% paraformaldehyde at room temperature for 10min, washing with PBS 3 times, penetrating membrane with 1% Triton X-100 for 10min, washing with PBS 3 times, and sealing with goat serum for 40 min. One group is added with monoclonal antibody SOX-2 (diluted by 1: 100), the other group is added with Ki-67 monoclonal antibody (diluted by 1: 100), the mixture is incubated overnight at 4 ℃, PBS is washed for 3 times, the two groups are respectively added with FITC marked goat anti-mouse IgG (diluted by 1: 100), the mixture is incubated for 1h at room temperature in dark, PBS is washed for 3 times, DAPI staining is carried out for 10min, PBS is washed for 3 times, mounting medium is mounted, and observation and photographing are carried out under a laser confocal microscope. The results of cell immunofluorescence detection of Ki-67, SOX-2, DELTA-CATENIN, and CLAUDIN-1 protein expression are shown in FIG. 5.

4. Freezing and recovering cells: after cell cryopreservation and recovery, the cells grow well and can be continuously passaged. And (3) detecting microbial contamination: the cells are free of bacterial, fungal or mycoplasma contamination.

5. Cell counting: and wiping the counting plate and the cover plate and covering the counting plate with the cover plate. After digesting the cells conventionally, sucking a little of the cell suspension, dripping the cell suspension on the edge of the cover slip to enable the suspension to fill the space between the cover slip and the counting plate, standing for 3min, paying attention to no air bubbles below the cover slip, and enabling the suspension not to flow into a side groove. The total number of cells in the four large grids of the plate is counted, and the line pressing cells are counted only on the left side and the upper side. Then, calculating according to the formula: cell number/mL-total of four large cells/4 × 10⁴。

The obtained cell line is named as a canine breast cancer cell line and is recorded as canine milk F17, and is preserved in China general microbiological culture Collection center (CGMCC) at 26.05.18 years, the preservation unit is abbreviated as CGMCC, the preservation number is CGMCC No.22349, and the preservation unit address is No. 3 of Beijing Kogyang district Beichen West Lu No. 1.

Example 2 Balb/SCID nude mouse model of canine mammary cancer

In order to obtain a good mouse xenograft model, 10 obtained after cell counting in example 1 was used⁸Individual canine milk F17 cell suspensions (resuspended in 0.2ml PBS) were subcutaneously inoculated into left mammary fat pads of 10 5-week-old female Balb/SCID nude mice (purchased from the center of the beijing vintonia test animal). Mice were observed weekly for tumor development. When tumors were found, the length and width of the tumors were monitored weekly with calipers, for example as shown in fig. 3, for 8 weeks. Mice were sacrificed when they developed dyspnea, weakness, or no obvious tumor. Tumors and organs were collected and histopathologically examined in 4% paraformaldehyde (pH 7.4). All the items are completed according to the 'instruction for nursing and using experimental animals'. The result shows that the tumor formation rate of the CMT-1 cell line nude mouse inoculated subcutaneously is 100 percent, the nude mouse invades and infiltrates subcutaneous and dermal layers, and deep layers of tumor are necrotized. After 4 weeks, spontaneous lung metastasis rate was 100%, and histopathological examination confirmed metastatic adenocarcinoma. The tumor growth curve of the transplanted tumor model conforms to a Gompertz function model and grows in a logarithmic mode after a latency period.

The specific operation is as follows:

1. and (3) conventional cell culture:

when the CMT-1 culture reached 90% confluency, it was washed 3 times with PBS, then the cells were digested with 0.25% trypsin (HyClone, USA) for 5min, the digestion was stopped by adding complete medium containing 15% serum, centrifuged at 1000r/min for 5min, the cells were collected, the supernatant was discarded, fresh medium was added, and the blown-down cells were replaced with new 25cm cells²In bottle, concentration is 1x10⁵cells/ml. Denoted CMT-1. And (5) carrying out 1 passage every 3d, wherein the seed separation rate is 1: 3.

Establishing a Balb/SCID nude mouse tumor-bearing model:

to obtain a good mouse xenograft model, cells were cultured to 90%, digested with 0.25% trypsin for 5min, digested by adding complete medium containing 15% fetal bovine serum 15%, centrifuged at 1000r/min for 5min, cells were collected and counted at 10⁸Individual canine milk F17 cell suspensions (resuspended in 0.2ml PBS) were each subcutaneously inoculated into the left mammary fat pad of 10 5-week-old female Balb/SCID nude mice. Mice were observed weekly for tumor development. Mice were sacrificed when they developed dyspnea, weakness, or no obvious tumor. Tumors and organs were collected and histopathologically examined in 4% paraformaldehyde (pH 7.4).

Experimental observation and verification are carried out, and the tumor-bearing mice have the following characteristics:

the tumor formation rate is as follows: the dog milk F17 cells are inoculated to Balb/SCID nude mice for 100 percent tumorigenesis, and the tumor growth accords with a Gompertzian index function model.

Tumor formation time: subcutaneous lumps at the original injected cells were observed one week after inoculation of canine milk F17 cells, and had a hard and pink texture.

Tumor growth conditions: the exponential proliferation phase is at weeks 2-5.

3. Tumor and cancer metastasis in tumor-bearing mice:

tumor cells infiltrating into subcutaneous and dermal layers of the breast inoculation part can be seen, and the deep layer of the tumor is necrotic. Obvious glandular epithelial cell invasion, sheet distribution, space occupation, compaction and central necrosis of a large-area tumor cell infiltration area can be seen in lung tissues. Therefore, the filial cells of the canine breast cancer cell line basically or completely retain the characteristics of the parent cells and have higher stability. The in-situ inoculation tumor formation rate and the spontaneous lung metastasis rate are 100 percent, the clinical characteristics of the triple negative breast cancer are reproduced, the model is an ideal and reliable research model, is suitable for in-vitro and in-vivo research on the canine triple negative breast cancer, and can provide a good in-vitro test model for the research on tumor occurrence development mechanism, drug target point and drug treatment mechanism.