CN109837250B

CN109837250B - Immortalized cell line of lamb testicular supporting cell and establishment method and application thereof

Info

Publication number: CN109837250B
Application number: CN201910087613.6A
Authority: CN
Inventors: 张志东; 王光祥; 王艳华; 秦晓东; 李彦敏; 杨能能; 贾宁; 苏雅杰
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2019-01-15
Filing date: 2019-01-29
Publication date: 2021-10-12
Anticipated expiration: 2039-01-29
Also published as: CN109837250A

Abstract

The invention discloses a lamb testicular supporting cell immortalized cell line and an establishing method and application thereof. The lamb testicular supporting cell immortalized cell line is named as a lamb testicular supporting cell immortalized cell line hTERT-LSC, and the preservation number is CCTCC NO: C2018202. experiments show that the hTERT-LSC cell line can be continuously passed for more than 60 generations, the growth and proliferation characteristics are kept unchanged, the cell line is sensitive to the inoculation capripoxvirus, the capripoxvirus can be efficiently proliferated, and the uniformity and the stability of the virus can be ensured. The titer of the capripoxvirus proliferated by the cell line is equivalent to that of the lamb testis supporting cell LSC, the cell seed separation rate is higher than that of the LSC, and the seed separation rate can reach 1: 3. Meanwhile, the immortalized cell line is used for preparing the vaccine, thereby simplifying the production process, shortening the production period, reducing the production cost and simultaneously ensuring the stable quality of the prepared capripox virus vaccine. Therefore, the invention provides a new technical means for the large-scale production of the capripoxvirus vaccine.

Description

Immortalized cell line of lamb testicular supporting cell and establishment method and application thereof

Technical Field

The invention relates to an immortalized cell line and an establishment method and application thereof, in particular to a lamb testis supporting cell immortalized cell line and an establishment method and application thereof in multiplication culture of capripoxvirus. The invention belongs to the technical field of cell engineering.

Background

Testicular Support Cells (SC) are epithelial cells on the basement membrane of the seminiferous tubule that provide support, energy, and nutrition for the differentiation and maturation of the spermatogenic cells, known as "nurse cells" of spermatogenic cells. In the process of differentiation and maturation of sperms, the supporting cells not only transmit gonadotropins to spermatogenic epithelium and control a series of dynamic processes of mitosis, meiosis and subsequent differentiation of spermatogenic cells, but also have multiple functions of supporting, nourishing, phagocytizing, releasing, secreting and the like on the sperms. Studies have shown that testicular support cells can secrete Androgen Binding Protein (ABP), Mullerian Inhibiting Substance (MIS), inhibin (inhibin), activin (activin), transforming growth factor beta (TGF β), insulin-like growth factor 1(IGF1), interleukins 1 and 6(IL1, IL6), Spermatogenic Growth Factor (SGF), neurotrophin 3(neurotrophin3, NT3), nerve growth factor (nerve growth factor, NGF), plasminogen activation, etc. to support proliferation and differentiation of spermatogonium.

Capripoxvirus (CPV) is the most important animal poxvirus, members of the genus Capripoxvirus (capropxvirus) belonging in the family Poxviridae (Poxviridae) the subfamily chordopoxvirinae (chordopoxriae) on a taxonomic level, including Capripoxvirus (gatporx virus, GTPV), Sheeppoxvirus (SPPV) and bovine sarcoidosis virus (Lumpy skin disease virus, LSDV). Capripox is an acute and contact infectious disease caused by capripox virus, is listed as one of 93 animal epidemic diseases which must be reported by the world animal health organization (FAO/OIE), and is listed as an infectious disease in China. The morbidity can reach more than 75%, the mortality can reach more than 50%, the mortality of lambs can reach 100%, and meanwhile, the abortion of ewes can also be caused. Capripox is one of the ancient diseases, and Hansen reported norway in 1879 for the first time. Later, the disease was reported in other parts of the world in succession. The disease is widely distributed in Africa, the middle east, the Indian subcontinent, northern Europe, Mediterranean countries, Germany, Australia, the United states and the like, and is more prevalent in particular in parts of the North Africa, the middle east and Asia. The surrounding countries bordering China, such as India, Bengal, Nibor, Pakistan, Russia, Mongolia, etc., have the prevalence of the disease. In recent years, Jiangsu, Guangdong, Guizhou, Shandong, Zhejiang, Guangxi, Gansu, Heilongjiang and other places in China all have the prevalence of the disease. The outbreak and epidemic of the disease cause the productivity of livestock to be reduced, the trade of live livestock and livestock products is stopped, and great economic loss is caused to the development of sheep raising industry.

The rapid and effective pathogen isolation for viral infectious diseases is the key of diagnosis and epidemic disease prevention and control work. The cells (lines, strains) capable of efficiently propagating the virus are one of the most important tools for virus diagnosis and subsequent research. To prevent the capripox from flooding and epidemic in China, the immunization of the capripox vaccine is the only effective measure for preventing the capripox. Capripoxvirus (GTPV, SPPV) can grow on primary cells of testis Caprae seu Ovis, kidney cells, BHK21 and Vero cells and produce cytopathic effect (CPE), but the virus titer is low, and especially the virus titer required by preparing vaccine by passage cells (such as BHK21 and Vero cells) can not be achieved. In China, the goatpox attenuated vaccine is produced by using primary cells of newborn goat testes, the manufacturing process is complex and laggard, the primary cells of the newborn goat testes are needed for breeding the goatpox attenuated antigen, but the preparation process of the primary cells is complex, the production cost is too high, the production efficiency is low, the inconvenience is brought to the large-scale production of the goatpox vaccine in a vaccine factory, and enterprises are difficult to obtain rich profits. Meanwhile, the newborn sheep testes used for reproducing the sheep pox attenuated antigens are different in variety every time, so that the quality of the produced attenuated vaccine is difficult to keep stable, and in addition, the newborn sheep testes have potential risks of carrying other pathogens. Therefore, the development of a continuous cell line suitable for the proliferation of the capripoxvirus has certain practical significance for vaccine enterprises.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a method for separating and purifying primary cells of lamb testis to obtain lamb testis supporting cells (LSC), a liposome transfection method is adopted to introduce eukaryotic expression plasmids pCI-neo-hTERT for coding hTERT and neo genes into the lamb testis supporting cells (LSC), and the LSC cells are immortalized and transformed to obtain the immortalized cell line of the lamb testis supporting cells.

In the present invention, preferably, the said immortalized cell line of lamb testis supporting cell is named as hTERT-LSC, and classified as hTERT-LSC, which is preserved in China center for type culture Collection, and the address is in China, Wuhan, the preservation number is CCTCC NO: c2018202, with a preservation time of 2018, 10 months and 12 days.

The invention relates to a lamb testicular supporting cell immortalized cell line which is prepared by the following steps:

(1) healthy male lambs born by healthy female lambs are selected, after slaughter, testicular parenchyma is collected aseptically, and primary cells of the lambs testicles are prepared by a collagenase and pancreatin mixed enzyme (0.1 wt% collagenase type IV (GIBCO), 0.25 wt% pancreatin (GIBCO)) digestion method. Adopting 6h differential adherence, 50mmol/L Tris-HCl to perform hypotonic treatment, high temperature culture separation at 38.5 ℃, purification and identification on the cultured primary cells of the testis of the lamb, and obtaining the testis supporting cells (LSC) of the lamb with the purity of more than 95 percent.

(2) Eukaryotic expression plasmids pCI-neo-hTERT encoding hTERT and neo genes are respectively introduced into separated and purified lamb testis supporting cells (LSC) by adopting a liposome transfection method, and G418 with the concentration of 350 mug/mL is screened. Obtaining G418-resistant positive clones, extracting RNA from screened G418-resistant positive cell clone (hTERT-LSC) cells, and detecting by using an RT-PCR method to find that the G418-resistant positive cell clones of the detection generations all express the hTERT gene. The successful transfer of the hTERT gene into the LSC cells is shown. And the effective expression is obtained, and the G418 resistant positive cells obtained by screening are named as hTERT-LSC.

(3) Transferring the hTERT-LSC cells to a 96-well plate by a limiting dilution method, picking a cloning well in which only a single cell grows, and then carrying out amplification culture until a monoclonal hTERT-LSC cell strain is obtained.

(4) And (4) carrying out cell biological characteristic determination on the monoclonal hTERT-LSC cell strain obtained by screening.

Respectively inoculating screened hTERT-LSC monoclonal cell strains into 6-hole plates, and observing the cell seed separation rate and the cell full monolayer time; GTPV cells are inoculated for strong toxicity, and CPE generation of each monoclonal cell strain is observed. Experiments prove that the hTERT-LSC monoclonal cell strain has the seed separation rate of 1:3, the full monolayer growth time of about 48 hours, and the time required for inoculating GTPV cells with strong toxicity of 75% to generate CPE is about 72 hours.

Wherein, the screened hTERT-LSC monoclonal cells 1# and 2# are subjected to continuous passage, and the time for the cells to grow full of a monolayer in each passage is recorded. The GTPV cell virulent virus (lgTCID) of the same generation is inoculated every 5 generations of the hTERT-LSC monoclonal cell strain₅₀≧ 5.0), observing CPE of GTPV to hTERT-LSC monoclonal cell strain, and determining TCID₅₀. Experiments prove that the cell strains of the hTERT-LSC monoclonal cells 1# and 2# still keep vigorous activity when the cells are passaged to 60 generations. And has high multiplication capacity on GTPV virus, and the cell produces CPE regularly and has virus titer (lgTCID)₅₀) Are all above 5.0. Therefore, the hTERT-LSC immortalized cell line is successfully established and can efficiently proliferate capripoxvirus.

The hTERT-LSC monoclonal cell strain obtained by screening is detected by an MTT method to find that hTERT-LSC 1#, 2# and LSC cells can not grow in a serum-free culture medium, and cells begin to become round, float and die at the 2 nd after liquid change. The proliferation promoting effect of 50ml/L FBS is weaker, while the proliferation promoting effect of 100ml/L FBS and 200ml/L FBS can obviously promote the proliferation of two cells (p is less than 0.05), wherein the proliferation promoting effect of 200ml/L FBS on hTERT-LSC is more obvious than that of 100ml/L FBS, and the difference between the two is obvious (p is less than 0.05). This suggests that hTERT-LSC is not significantly altered from LSC in serum dependence.

Wherein LSC and hTERT-LSC cells in the logarithmic growth phase are taken, colchicine is added to the cells until the concentration is 0.1 mu g/m L, and the cells are continuously cultured for 4-6 h. Cells were trypsinized at 2.5g/L, centrifuged at 1000r/min for 5min and the supernatant discarded. 50mmol/L KCl resuspended cell pellet and hypotonic treated at room temperature for 60 min. Then adding methanol/glacial acetic acid (3:1) fixing solution for 30min, re-suspending, centrifuging at 4 ℃, and discarding the supernatant. Continuously adding the fixing solution, re-suspending and uniformly mixing, dropwise adding the cell suspension on a clean glass sheet precooled at 4 ℃, and drying in air. Staining with Jiemsa for about 10min, dehydrating and sealing, and observing the chromosome banding pattern under oil microscope. Both hTERT-LSC and LSC cells have 42 chromosomes; and is still diploid cell, and the chromosome morphological structure is normal.

The 60 th generation hTERT-LSC 1#, 2# immortalized monoclonal cell strain and 17 th generation LSC cell are used as test cell group, and simultaneously, Hela cell is used as positive control group, CEF (chicken embryo fibroblast) is used as negative control group, and nude mouse inoculation test is carried out. Experiments prove that after the immortalized monoclonal cell strains of hTERT-LSC 1#, 2# and the LSC 17 generation are inoculated to the nude mice, the nude mice have no adverse symptoms and no tumor on the back in the experimental period. The materials are taken 2 months after inoculation, and histological observation shows that the tissue structure of the inoculated area is not obviously different from that of the uninoculated part. The nude mice of the live cell group inoculated with Hela cells showed a tumor at the inoculated site 7 days after the inoculation, and the tumor size was about 10mm 30 days after the inoculation. The histopathological section examination shows that the cells are disorganized, the polarity disappears, and a large number of nuclear fission phases can be seen. The hTERT-LSC1 and LSC cells were proved to be non-tumorigenic.

Furthermore, the invention also provides application of the lamb testis supporting cell immortalized cell line in culturing and proliferating Capripoxvirus (CPV).

Compared with the prior art, the invention has the beneficial effects that:

the invention successfully introduces the human telomerase reverse transcriptase gene into the LSC cell, activates the telomerase activity and prolongs the service life of the cell cultured in vitro. According to the invention, experiments show that the newborn bovine testis supporting cell immortalized cell line hTERT-LSC can be continuously passed for more than 60 generations, and the growth and proliferation characteristics are kept unchanged. The cell line is sensitive to the breeding of the capripoxvirus, can efficiently proliferate the GTPV virus, and can ensure the uniformity and stability of the virus. The virus titer proliferated by the cell line is equivalent to that of the lamb testicular supporting cell LSC, the cell seed separation rate is higher than that of the LSC, and the seed separation rate can reach 1: 3. Meanwhile, the immortalized cell line is used for preparing the vaccine, thereby simplifying the production process, shortening the production period, reducing the production cost and simultaneously ensuring the stable quality of the prepared capripox virus vaccine. Therefore, the invention provides a new technical means for the large-scale production of the capripoxvirus vaccine.

Drawings

FIG. 1 shows the isolation and purification of cultured lamb testis supporting cells (LSCs);

wherein, A: culturing the support cells for 0 hour; b: culturing the support cells for 2 h; c: culturing the support cells for 12 h; d: culturing the support cells for 72 h;

FIG. 2 is the identification of testicular support cells from a lamb;

wherein, A: third generation HE staining of support cells; b: the third generation supports the detection of cell FasL protein; c: detecting Overlay by using a third generation of support cell FasL protein; d: a negative control for detecting FasL protein of MDBK cells;

FIG. 3 is a schematic diagram of the RT-PCR detection of marker genes in support cells;

wherein, 1. SCF; GDNF; DL2000 Marker;

FIG. 4 is a map of the pCI-neo-hTERT expression vector;

FIG. 5 shows hTERT-LSC positive clones;

FIG. 6 shows RT-PCR detection of hTERT gene for positive cloning of hTERT-LSC cells;

wherein, M is DL2000 marker; 1, hTERT gene positive control;

FIGS. 7A-F are selected monoclonal cells of a portion of hTERT-LSC;

FIG. 8 is the serum dependence of hTERT-LSC and LSC cells;

FIGS. 9A and B show pathological sections of tumors from mice inoculated with Hela cells (HE staining 400X).

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

EXAMPLE 1 isolation and purification of cultured lamb testis support cells (LSC)

1.1 separation, purification and culture of testis supporting cells (LSC) of lamb

Healthy male lambs born by healthy ewes (with healthy constitution and negative foot-and-mouth disease, brucellosis and tuberculosis detection) are selected, scrotum (the root of the scrotum is ligated and the outside of the scrotum is disinfected by 75% alcohol) is collected after slaughter, and the scrotum is returned to a laboratory in a constant temperature box within 2 hours. Testicular parenchyma were aseptically harvested and primary lamb testicular cells were prepared by digestion with collagenase and pancreatin mix (0.1 wt% collagenase type IV (GIBCO), 0.25 wt% pancreatin (GIBCO)).

Adopting 6h differential adherence, 50mmol/L Tris-HCl to perform hypotonic treatment on the cultured primary cells of the lamb testis, culturing at high temperature of 38.5 ℃, separating and purifying to obtain the lamb testis supporting cells (figure 1).

1.2 identification of testis support cells (LSCs) in lamb

HE staining is carried out on the lamb testis supporting cell line separated, purified and cultured in section 1.1 to observe the cell morphology, and meanwhile, immune histochemical identification is carried out on the lamb testis supporting cell by adopting FasL protein.

The immunohistochemical method was as follows: fixing the cultured 3d lamb testis supporting cells with 4% paraformaldehyde solution for 10min, rinsing with PBS for 3 times multiplied by 5min, carrying out closed incubation for 20min at room temperature on goat serum, adding a rabbit anti-rat FasL polyclonal antibody (abcam, diluted 1: 100) primary antibody mixed working solution, incubating for 3h at 37 ℃, rinsing with PBS for 3 times, adding FITC-labeled goat anti-rabbit IgG (abcam, diluted 1: 50) secondary antibody mixed working solution, incubating for 30min at 37 ℃, rinsing with PBS for 3 times, randomly selecting 10 visual fields under a fluorescence microscope, and observing the dyeing result of FasL under green fluorescence. And (4) calculating the total number of FasL positive cells and cells in each visual field, and calculating the percentage of the positive cells to obtain the purity of the lamb testis supporting cells.

Extracting total RNA of the testicular support cells of the lambs, detecting the expression conditions of marker genes SCF and GDNF of the support cells after reverse transcription (the primer sequences are shown in Table 1), and the reaction conditions are as follows: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 58 ℃ for 30s, 72 ℃ for 20 s; 7min at 72 ℃. The primers were synthesized by Dalibao organism (TaKaRa) Co., Ltd.

TABLE 1 primer sequence information

By HE staining, it can be seen that the obtained testis supporting cells of the lamb have irregular shapes, the cytoplasm is completely spread out, the staining is lighter, the nucleus is stained more deeply, the round or oval shape is positioned in the center or the deviation of the cytoplasm, and the nucleolus is obvious (figure 2A); the indirect immunofluorescence detects that the testicular supporting cells express the FasL protein, and as a result, the supporting cells which are separated, purified and cultured almost see green fluorescence (figure 2C, D), and the positive rate of the supporting cells reaches more than 95%. No green fluorescence was detected from MDBK cell negative control cells (fig. 2B). The RT-PCR results show that: support the significant expression of the marker genes GDNF and SCF in cells (figure 3).

EXAMPLE 2 establishment of immortal cell line of lamb testis supporting cells (hTERT-LSC)

2.1 pCI-neo-hTERT plasmid transfection of LSC cells

The method comprises the steps of separating and purifying primary cells of the lamb testis in example 1 to obtain lamb testis supporting cells, introducing eukaryotic expression plasmids pCI-neo-hTERT (a vector map is shown in figure 4) for coding hTERT and neo genes into LSC (generation 17) according to the requirements of a Lipofectamine 2000Regent transfection instruction of Invitrogen company, and carrying out immortalization transformation on an LSC-limited continuous cell line.

2.2 determination of the optimal screening concentration for neomycin (G418)

LSC (passage 17) cells were prepared as a cell suspension at 1X 10⁵one/mL was inoculated into a 24-well plate. G418 was applied to each well in a gradient of 50. mu.g/mL over a range of 100. mu.g/m L-1000. mu.g/mL, with different concentrations of selection medium. Each gradient was replicated 4 wells. According to the growth of the cells, every 2d is more liquid. The minimum G418 concentration that kills all cells within 10d of screening is the optimal screening concentration.

Experiments demonstrated that the lowest concentration at which seeded LSC cells were found to die overall was 350. mu.g/mL. This concentration was chosen as the immortalized cell in this experiment: optimal screening concentration of hTERT-LSC.

2.3 acquisition of hTERT-LSC Positive cells

When 80% of LSC cells transfected with pCI-neo-hTERT plasmids are converged, discarding culture supernatant, replacing new cell culture solution and adding G418 to make the final concentration of the cell culture solution 300 mu G/mL, continuously culturing for two weeks, and replacing G418 cell culture solution with the same concentration every 2 d; meanwhile, LSC untransfected cells are used as blank control, and the growth and death conditions of the cells are observed day by day. Until positive clones were visible. The anti-G418 positive clone cells were expanded to obtain anti-G418 positive clones (FIG. 5). And (3) extracting RNA from the screened anti-G418 positive clone cells, and detecting the expression of exogenous hTERT in the transfected cells by using an RT-PCR method.

As shown in FIG. 6, specific fragments of about 400bp were amplified in two generations of anti-G418-positive clone cells, and the length of the fragments was consistent with that expected. And (3) screening positive cells to express the hTERT gene, indicating that the hTERT gene is successfully transferred into the LSC cells and effectively expressed, and the G418 resistant positive cells obtained by screening are named as hTERT-LSC.

2.4 hTERT-LSC monoclonal cell screening

Transfer the hTERT-LSC (16 generation) cells to a 96-well plate by a limiting dilution method, pick out a clone well in which only a single cell grows, and then expand and culture the clone well to a 24-well plate and a 6-well plate. A16 hTERT-LSC monoclonal cell strain (FIG. 7) was obtained and sequentially named as hTERT-LSC 1# -16 #.

EXAMPLE 3 hTERT-LSC biological Property assay

3.1 hTERT-LSC monoclonal cell growth characteristics and viral proliferation

The screened hTERT-LSC monoclonal cell strains 1# -16 # in the example 2 are respectively inoculated into a 6-well plate, the seed separation rate (which means how many cell bottles with the same volume can be inoculated with primary cells in 1 cell bottle for subculture) is observed, and the cells grow for a monolayer time; inoculating the full monolayer hTERT-LSC monoclonal cell strain No. 1-16 # with GTPV cell virulent strain (lgTCID)₅₀Not less than 6.0), and observing the CPE generation of the virus on the cells, and the result is shown in table 2.

TABLE 2 hTERT-LSC monoclonal cell growth characteristics and viral proliferation profile

As can be seen from Table 2, each monoclonal cell strain of hTERT-LSC has stable growth characteristics, the growth speed and the cell seed separation rate are consistent, a monolayer grows after 48 hours, CPE is generated on GTPV cells in a relatively regular way, and the time for generating 75% of CPE by the cells is maintained at about 72.

3.2 passage of hTERT-LSC cells and its proliferation assay for GTPV

The selected hTERT-LSC monoclonal cells 1# and 2# in example 1 were serially passaged and the time for the cells to grow full of a monolayer was recorded for each passage. The GTPV cell virulent virus (lgTCID) of the same generation is inoculated every 5 generations of the hTERT-LSC monoclonal cell strain₅₀≧ 5.0), observing CPE of GTPV to hTERT-LSC monoclonal cell strain, and determining TCID₅₀。

Experiments prove that when the hTERT-LSC monoclonal cells 1# and 2# are passaged to 60 generations, cell strains still keep vigorous activity, high multiplication capacity on GTPV virus is kept, CPE (CPE) generated by the cells is relatively regular, and the virus titer (lgTCID) is obtained₅₀) Above 5.0, the results of passage of hTERT-LSC monoclonal cell No. 1 and its proliferation to GTPV are shown in Table 3. Thus, the establishment of the hTERT-LSC immortalized cell line was successful.

TABLE 3 passage of hTERT-LSC monoclonal cell # 1 and its proliferation profile to GTPV

3.3 hTERT-LSC monoclonal cell serum dependency

The MTT method is used for detecting the serum dependence of cells and comprises the following specific steps:

(1) hTERT-LSC 1#, 2# and 17 th generation LSC cells at passage 58 were 1X 10⁴The individual cells/well were seeded in 96-well culture plates.

(2) 24h after inoculation, the cell fluid was replaced with DMEM/F12 medium containing 50m L/L FBS, 100m L/L FBS, and 200m L/L FBS, respectively. For each gradient, 3 replicate wells were made, with 3 blank wells.

(3) Incubating for 14h in an incubator with saturated humidity and 5% CO2 at 37 ℃.

(4) 10 μ L of MTT (10mg/m L) reaction solution was added and incubation was continued at 37 ℃ for 2-4 h until visible needle-shaped purple crystals under the microscope.

(5) Add 100. mu.L DMSO to each well, including blank wells, and mix gently.

(6) And (3) keeping the light away for 2-4 h, and measuring the light absorption value on an enzyme-linked immunosorbent assay instrument, wherein the measurement wavelength is 490 nm. Zeroed with blank wells and plotted.

The hTERT-LSC 1#, 2# and LSC cells can not grow in a serum-free culture medium, and cells begin to become round, float and die at the 2 nd day after liquid change. The proliferation promoting effect of 50ml/L FBS is weaker, while the proliferation promoting effect of 100ml/L FBS and 200ml/L FBS can obviously promote the proliferation of two cells (p is less than 0.05), wherein the proliferation promoting effect of 200ml/L FBS on hTERT-LSC is more obvious than that of 100ml/L FBS, and the difference between the two is obvious (p is less than 0.05). This suggests that hTERT-LSC is not significantly altered from LSC in serum dependence (FIG. 8).

3.4 hTERT-LSC and LSC cell karyotype analysis

Taking hTERT-LSC and LSC cells in the logarithmic growth phase, adding colchicine to the concentration of 0.1 mu g/m L, and continuously culturing for 4-6 h. Cells were trypsinized at 2.5g/L, centrifuged at 1000r/min for 5min and the supernatant discarded. 50mmol/L KCl resuspended cell pellet and hypotonic treated at room temperature for 60 min. Then adding methanol/glacial acetic acid (3:1) fixing solution for 30min, re-suspending, centrifuging at 4 ℃, and discarding the supernatant. Continuously adding the fixing solution, re-suspending and uniformly mixing, dropwise adding the cell suspension on a clean glass sheet precooled at 4 ℃, and drying in air. Staining with Jiemsa for about 10min, dehydrating and sealing, and observing the chromosome banding pattern under oil microscope. Both hTERT-LSC and LSC cells have 42 chromosomes; and is still diploid cell, and the chromosome morphological structure is normal.

3.5 nude mouse tumorigenic test

A58 th generation hTERT-LSC 1#, 2# immortalized monoclonal cell strain and a 17 th generation LSC cell are used as a test cell group, and meanwhile, a naked mouse inoculation test is carried out by taking Hela cells as a positive control group and CEF (chicken embryo fibroblast) as a negative control group. All cells used in the test were prepared in duplicate for the same passage, and were divided into viable cell group and cell freeze-thaw lysis group (cell freeze-thaw lysis group, which was repeatedly frozen and thawed 3 times before the test to ensure complete lysis of all cells). Each group of samples is inoculated with 10 nude mice, each of the males and the females is half, each sample is inoculated with 2 groups of nude mice, wherein, 1 group of living cells and 1 group of freeze-thaw cells are inoculated, the subcutaneous cell suspension is inoculated at 1/3 positions behind the neck or in front of the back of each nude mouse, the inoculation dose is 0.2 ml/mouse, and the cell concentration is 10⁶～10⁷/0.2ml。

After inoculation of cell suspension (live cells and freeze-thaw disrupted cells), nude mice were observed day by day and recorded for mental, dietary, bowel movements and tumor growth, and the length and width of the tumor mass and nodules were measured with a vernier caliper. After 14 days of observation, the presence or absence of nodules or tumor formation was examined. If there is a nodule or suspicious lesion, the neck should be cut off and killed, dissected and examined for 1-2 weeks for histopathological observation. And observing half of the nude mice without the nodule or suspicious lesion for 12 weeks, performing autopsy, observing the other half of the nude mice after 12 weeks of neck fracture, performing autopsy, collecting each organ and lymph node of the nude mice, fixing by 10% glutaraldehyde, making pathological sections, and performing pathological histological observation.

After the immortalized monoclonal cell strains of hTERT-LSC 1#, 2# and the LSC 17 th generation are inoculated to the nude mice, the nude mice have no adverse symptoms and no tumor on the back in the experimental period. The material was taken 2 months after inoculation and histological observations showed no significant difference in the tissue structure of the inoculated area from the uninoculated site (table 4). The nude mice of the live cell group inoculated with Hela cells showed a tumor at the inoculated site 7 days after the inoculation, and the tumor size was about 10mm 30 days after the inoculation. The histopathological section examination revealed that the cells were disorganized, the polarity disappeared, and a large number of nuclear fission phases were visible (FIG. 9). Both the hTERT-LSC and LSC cell lines were demonstrated to be non-tumorigenic.

TABLE 4 results of tumor-inducing tests of hTERT-LSC and LSC on nude mice

In conclusion, the invention successfully introduces the human telomerase reverse transcriptase gene into the LSC cell, activates the telomerase activity of the LSC cell and prolongs the service life of the cell cultured in vitro. The lamb testis supports the immortalized cell line (hTERT-LSC) and still keeps the normal biological characteristics of the parent cell (LSC), and can efficiently proliferate GTPV virus. The cell line is delivered to the China center for type culture Collection of Wuhan university in 2018, 10 months and 12 days, and the preservation number is CCTCC NO: C2018202.

Claims

1. the immortalized cell line of the lamb testicular supporting cell is named as hTERT-LSC, is preserved in China center for type culture collection with the preservation number of CCTCC NO: c2018202, with a preservation time of 2018, 10 months and 12 days.

2. Use of a lamb testicular support cell immortalized cell line according to claim 1 for the culture or propagation of Capripoxvirus (CPV).