A giant panda immortalized kidney cell line GPK-1, preparation method and use thereof

Abstract

The present invention discloses a giant panda immortalized kidney cell line GPK‑1, a preparation method and a use thereof, and belongs to the field of immortalized cell technology. The preparation method is as follows: (1) collecting kidney tissue of a dead giant panda and immersing it in a protective solution; (2) performing two-step digestion on the kidney tissue treated in step (1); (3) culturing and propagating the digested cells, and then infecting the propagated cells with a lentivirus carrying a neomycin resistance gene and an SV40‑LT gene, and then screening and establishing a giant panda immortalized kidney cell line. The present invention provides a technology for efficiently isolating and culturing giant panda kidney-derived cells, and establishes an immortalized giant panda kidney-derived cell line GPK‑1. Compared with primary giant panda kidney cells, GPK‑1 retains normal karyotype and related molecular characteristics, and has been significantly improved in terms of cell passage times, proliferation efficiency and transfection efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of immortalized cells, and particularly relates to a giant panda immortalized kidney cell line GPK-1, a preparation method and uses thereof. Background Art

[0002] In the field of giant panda conservation research, the lack of research materials has become a bottleneck problem that hinders the in-depth development of related work. At present, the acquisition of giant panda research materials is mainly through the following channels, but they all face complex and diverse problems and challenges.

[0003] First of all, it is extremely difficult to obtain samples from living giant pandas under the premise of strictly following the principle of non-invasive collection. In order to ensure that the daily life of giant pandas is not disturbed, researchers can usually only collect non-damaging samples such as urine, feces, and hair. The application scenarios of these samples are limited and it is difficult to meet the needs of in-depth scientific research. Secondly, although rich tissue materials can be obtained from dead giant pandas and preserved for a long time through technical means such as fixation and freezing, this type of material is uncertain. Such resources are non-renewable resources and are extremely limited in quantity. Furthermore, on giant pandas, people have successfully isolated and cultured resources such as skin fibroblasts, milk-derived cells, and umbilical cord mesenchymal stem cells, providing valuable materials for scientific research on giant pandas. These cells will inevitably undergo aging during in vitro culture, making it difficult to maintain stability and repeatability of experimental results. Therefore, in order to meet the needs of a large number of experiments, these cells must be immortalized.

[0004] In the process of conducting research on major infectious diseases (such as parvovirus disease) and genetic diseases in giant pandas, establishing a stable immortalized kidney cell line is an important basis for conducting in vitro experiments. However, compared with model animals and domestic animals, the construction of immortalized cell lines for giant pandas faces the following two major technical difficulties. First, due to the particularity of giant pandas as endangered species, tissue samples can usually only be obtained through autopsy after the death of an individual. Since wild animal autopsies must follow strict operating procedures, it often takes more than 6 hours from the death of an individual to the completion of sample collection, causing the kidney tissue to experience long-term ischemia and hypoxia, seriously affecting the activity of primary cells and the success rate of culture. Secondly, the use of conventional kidney cell culture conditions cannot well culture giant panda kidney cells. The cells proliferate slowly and show obvious aging characteristics after 3-5 generations of culture, making it difficult to complete the key nodes of immortalization transformation. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a giant panda immortalized kidney cell line GPK-1, a preparation method and uses. The present invention provides a highly efficient separation and culture technology for giant panda kidney-derived cells, and establishes an immortalized giant panda kidney-derived cell line GPK-1. Compared with the primary kidney cells of the giant panda, GPK-1 retains the normal karyotype and related molecular characteristics, and has been significantly improved in terms of cell passage number, proliferation efficiency and transfection efficiency. In addition, the feasibility of GPK-1 as a cell model for giant panda disease research has been verified.

[0006] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0007] The purpose of the present invention is to provide a method for preparing a giant panda immortalized kidney cell line, comprising the following steps:

[0008] (1) Collect kidney tissue from a deceased giant panda and soak it in a protective solution for 10 to 20 minutes; the protective solution includes resveratrol, melatonin, and giant panda milk cell exosome extract;

[0009] (2) performing two-step digestion on the kidney tissue processed in step (1);

[0010] (3) The digested cells were cultured and subcultured, and then the subcultured cells were infected with a lentivirus carrying the neomycin resistance gene and the SV40-LT gene, and then the giant panda immortalized kidney cell line was screened and established.

[0011] Furthermore, the concentration of resveratrol in the protective solution was 50-80 μM, the concentration of melatonin was 30-50 μM, and the concentration of giant panda milk cell exosome extract was 50-100 μg / mL.

[0012] Furthermore, the preparation method of giant panda milk cell exosome extract is as follows:

[0013] Giant panda milk cells cultured for 1-5 generations were taken. When the cell confluence reached 80%, the complete medium was discarded. After washing the adherent cells, low-glucose DMEM without FBS was added and cultured for 48 h. The cell supernatant was collected and centrifuged to obtain the giant panda milk cell exosome extract.

[0014] Further, the specific process of the two-step digestion is:

[0015] Digest in water bath with digestion solution I at 37°C for 10-15 min. After centrifugation, continue digestion in water bath with digestion solution II for 10-15 min.

[0016] Further, the digestion solution I includes DMEM / F12, type IV collagenase at a concentration of 2-5 mg / mL, hyaluronidase at a concentration of 1-5 mg / mL, and DNaseI at a concentration of 0.1-0.5 mg / mL;

[0017] Digestion solution II includes DNaseI at a concentration of 0.1-0.5 mg / mL and 0.25% trypsin.

[0018] Furthermore, the process of culturing and subculturing the digested cells in step (3) is as follows:

[0019] The cells digested in step (2) were inoculated into a culture dish and cultured in DMEM high-glucose medium until the cell confluence reached 80%. After digestion and subculture, the cells were cultured in DMEM high-glucose modified medium for 72 to 96 hours.

[0020] DMEM high glucose modified culture medium includes DMEM high glucose, 10% FBS, 200~300 UI / mL penicillin, 200~260μg / mL streptomycin, 0.5~1.2μg / mL amphotericin, 10~20 ng / mL EGF, 10~20μM resveratrol and 50~80 mM trehalose.

[0021] Furthermore, the culture medium used in the screening in step (3) includes DMEM high glucose, 10% FBS, 10-15 ng / mL EGF, 10-15 ng / mL hydrocortisone, 10-15 μg / mL insulin, 10-12 μM resveratrol, 50-80 mM trehalose, and 800-1000 μg / mL G418.

[0022] Furthermore, the giant panda immortalized kidney cell line is the giant panda immortalized kidney cell line GPK-1, which was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on August 14, 2024, with the deposit number CCTCC NO: C2024252.

[0023] Another object of the present invention is to provide a giant panda immortalized kidney cell line GPK-1, which was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on August 14, 2024, with a deposit number of CCTCC NO: C2024252.

[0024] Another object of the present invention is to provide the use of the giant panda immortalized kidney cell line GPK-1 in drug screening or constructing an animal model.

[0025] Beneficial effects of the present invention:

[0026] The present invention has developed a complete set of isolation and culture methods for giant panda kidney primary cells, and cultivated an immortalized cell line GPK-1 that can be stably propagated. GPK-1 is in the form of epithelial cells and is the first giant panda epithelial-like cell line obtained so far. For giant pandas, whose research materials are extremely scarce, the establishment of the GPK-1 cell line provides an ideal in vitro research model for giant panda kidney disease, toxicological research, and drug screening, and provides important research support for the prevention and control of major epidemics and population safety of giant pandas.

[0027] Compared with the prior art, the present invention adopts a protective solution combined with a step-by-step (two-step) digestion method for treatment, which can effectively reduce the damage to cells caused by the enzyme digestion process, improve the viability of the obtained cells and the subsequent culture generations, thereby greatly improving the effects of virus infection and cell immortalization.

[0028] Compared with the existing technology, the present invention obtains the first immortalized giant panda kidney epithelial cell line, which can be stably and continuously cultured in vitro for more than 50 generations, solving the problem of scarcity of related resources and can be widely used in various subsequent scientific experimental research. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figures are morphological diagrams of giant panda kidney cells; Figure A is the 3rd generation cell morphology diagram of giant panda primary kidney cells; Figure B is the 10th generation cell morphology diagram of GPK-1; Figure C is the 30th generation cell morphology diagram of GPK-1; Figure D is the 50th generation cell morphology diagram of GPK-1;

[0030] Figure 2 The growth curves of giant panda primary kidney cells and GPK-1 at different generations;

[0031] Figure 3 The following are the results of the identification of molecular markers of giant panda kidney cells; Figure A is the results of immunofluorescence identification of giant panda primary kidney cells (generation 3); Figure B is the results of immunofluorescence identification of giant panda kidney cell line GPK-1 (generation 50); Figure C is the results of RT-PCR detection of giant panda primary kidney cells and GPK-1;

[0032] Figure 4 This is the result of karyotype analysis of giant panda kidney cell line GPK-1 (50th generation);

[0033] Figure 5 This is the detection result of feline parvovirus (FPV) infecting giant panda kidney cell line GPK-1. DETAILED DESCRIPTION

[0034] The specific implementation modes of the present invention are described below to facilitate those skilled in the art to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0035] The materials used in the present invention are as follows:

[0036] 1. The preparation method of giant panda milk cell exosomes is as follows:

[0037] Giant panda milk cells of 1-5 generations were cultured. When the cell confluence reached 80%, the complete medium was discarded, the adherent cells were washed twice with preheated PBS, and then low-glucose DMEM without FBS was added. After 48 h of culture, the cell supernatant was collected and exosomes were isolated by ultracentrifugation. The cell supernatant was centrifuged at 4 °C, 300 g for 10 min to remove dead cells; the supernatant was collected and centrifuged at 4 °C, 2000 g for 10 min to remove cell debris; the supernatant was transferred to a new centrifuge tube, centrifuged at 4 °C, 10, 000 g for 30 min to remove larger vesicles; and centrifuged at 4 °C, 100, 000 g for 70 min. The supernatant was removed and the exosome pellet was resuspended with 150 μL PBS and stored at -80 °C for later use.

[0038] 2. Melatonin and resveratrol were dissolved in DMSO to prepare a 10 mM stock solution, which was diluted when used.

[0039] 3. Conventional DMEM high glucose culture medium: DMEM high glucose, 10% FBS, 200 UI / mL penicillin, 200 μg / mL streptomycin, and 0.5 μg / mL amphotericin.

[0040] 4. Modified DMEM high glucose medium: DMEM high glucose, 10% FBS, 200-300 UI / mL penicillin, 200-260 μg / mL streptomycin, 0.5-1.2 μg / mL amphotericin, 10-20 ng / mL EGF, 10-20 μM resveratrol, and 50-80 mM trehalose;

[0041] Preferably, the modified DMEM high-glucose medium comprises: DMEM high-glucose, 10% FBS, 200 UI / mL penicillin, 200 μg / mL streptomycin, 0.5 μg / mL amphotericin, 10 ng / mL EGF (human source), 10 μM resveratrol, and 50 mM trehalose.

[0042] 5. Digestion solution I: DMEM / F12, 2-5 mg / mL type IV collagenase, 1-5 mg / mL hyaluronidase, and 0.1-0.5 mg / mL DNaseI; Digestion solution II: 0.1-0.5 mg / mL DNaseI and 0.25% trypsin.

[0043] Preferably, digestion solution I comprises: DMEM / F12, type IV collagenase at a concentration of 2 mg / mL, hyaluronidase at a concentration of 1 mg / mL, and DNaseI at a concentration of 0.1 mg / mL; digestion solution II comprises DNaseI at a concentration of 0.1~mg / mL, and 0.25% trypsin.

[0044] 6. Screening culture medium: DMEM high glucose, 10% FBS, 10~15ng / mL EGF, 10~15ng / mL hydrocortisone, 10~15μg / mL insulin, 10~12μM resveratrol, 50~80mM trehalose, and 800~1000 μg / mL G418;

[0045] The preferred screening medium includes: DMEM high glucose, 10% FBS, 10 ng / mL EGF, 10 ng / mL hydrocortisone, 10 μg / mL insulin, 10 μM resveratrol, 50 mM trehalose, and 800 μg / mL G418.

[0046] Example 1

[0047] A method for preparing a giant panda immortalized kidney cell line GPK-1, the specific process is as follows:

[0048] (1) Collect kidney tissue from the deceased giant panda and cut the cleaned kidney tissue into pieces of about 1 mm 3 The tissue blocks were cut into small pieces and then immersed in 10 mL of PBS solution containing 50 μM resveratrol, 30 μM melatonin and 50 μg / ml giant panda milk cell exosome extract for 10 min;

[0049] (2) Add the tissue blocks treated in step (1) to digestion solution I (components: DMEM / F12, 2 mg / ml type IV collagenase, 1 mg / ml hyaluronidase, 0.1 mg / ml DNaseI), digest in a 37°C water bath for 10 min, centrifuge at 60 g for 2 min and discard the supernatant; add digestion solution II (components: 0.25% trypsin, 0.1 mg / ml DNaseI), digest in a 37°C water bath for 10 min, and add an equal volume of fetal bovine serum to terminate the digestion;

[0050] (3) The digested primary cells (denoted as P1) were inoculated into a 6 cm culture dish and cultured with conventional DMEM high-glucose medium until the cell confluence was 80%. The cells were digested and passaged using 0.25% trypsin (denoted as P2). The P2 passage giant panda kidney cells were inoculated into a 24-well plate (inoculation density was 2 × 10 4 cells / well, and then cultured with modified DMEM high-glucose medium (DMEM high-glucose, 10% FBS, 200 UI / mL penicillin, 200 μg / mL streptomycin, 0.5 μg / mL amphotericin, 10 ng / mL EGF (human), 10 μM resveratrol, 50 mM trehalose) for 72 h to obtain P3 generation giant panda primary kidney cells;

[0051] (4) The P3 generation giant panda primary kidney cells were infected with lentivirus carrying the neomycin resistance gene and the SV40-LT gene for 2 h at an MOI of 5. The cells were then passaged at 2×10 4 The cells were seeded in 48-well plates at a concentration of 10 cells / mL, and 3 replicates were seeded in each way. After 48 h of culture, the cells were treated with screening medium (DMEM high glucose, 10% FBS, 10 ng / mL EGF, 10 ng / mL hydrocortisone, 10 μg / mL insulin, 10 μM resveratrol, 50 mM trehalose, and 800 μg / mL G418) for 24 h to obtain the giant panda immortalized kidney cell line GPK-1.

[0052] Example 2

[0053] A method for preparing a giant panda immortalized kidney cell line GPK-1, the specific process is as follows:

[0054] (1) Collect kidney tissue from the deceased giant panda and cut the cleaned kidney tissue into pieces of about 1 mm 3 The tissue blocks were cut into small pieces and then immersed in 10 mL of PBS solution containing 60 μM resveratrol, 40 μM melatonin and 80 μg / ml giant panda milk cell exosome extract for 10 min;

[0055] (2) Add the tissue blocks treated in step (1) to digestion solution I (components: DMEM / F12, 3.5 mg / ml type IV collagenase, 2.5 mg / ml hyaluronidase, 0.3 mg / ml DNaseI), digest in a 37°C water bath for 10 min, centrifuge at 60 g for 2 min and discard the supernatant; add digestion solution II (components: 0.25% trypsin, 0.3 mg / ml DNaseI), digest in a 37°C water bath for 10 min, and add an equal volume of fetal bovine serum to terminate the digestion;

[0056] (3) The digested primary cells (denoted as P1) were inoculated into a 6 cm culture dish and cultured with conventional DMEM high-glucose medium until the cell confluence was 80%. The cells were digested and passaged using 0.25% trypsin (denoted as P2). The P2 passage giant panda kidney cells were inoculated into a 24-well plate (inoculation density was 2 × 10 4 cells / well, and then cultured with modified DMEM high-glucose medium (DMEM high-glucose, 10% FBS, 240 UI / mL penicillin, 220 μg / mL streptomycin, 1.2 μg / mL amphotericin, 15 ng / mL EGF (human), 14 μM resveratrol, 60 mM trehalose) for 72 h to obtain P3 generation giant panda primary kidney cells;

[0057] (4) The P3 generation giant panda primary kidney cells were infected with lentivirus carrying the neomycin resistance gene and the SV40-LT gene for 2 h at an MOI of 5. The cells were then passaged at 2×10 4 The cells were seeded in 48-well plates at a concentration of 10 cells / mL, and 3 replicates were seeded in each way. After 48 h of culture, the cells were treated with screening medium (DMEM high glucose, 10% FBS, 12 ng / mL EGF, 12 ng / mL hydrocortisone, 12 μg / mL insulin, 11 μM resveratrol, 60 mM trehalose, and 860 μg / mL G418) for 24 h to obtain the giant panda immortalized kidney cell line GPK-1.

[0058] Example 3

[0059] A method for preparing a giant panda immortalized kidney cell line GPK-1, the specific process is as follows:

[0060] (1) Collect kidney tissue from the deceased giant panda and cut the cleaned kidney tissue into pieces of about 1 mm 3 The tissue blocks were cut into small pieces and then immersed in 10 mL of PBS solution containing 80 μM resveratrol, 50 μM melatonin and 60 μg / ml giant panda milk cell exosome extract for 10 min;

[0061] (2) Add the tissue blocks treated in step (1) to digestion solution I (components: DMEM / F12, 5 mg / ml type IV collagenase, 5 mg / ml hyaluronidase, 0.5 mg / ml DNaseI), digest in a 37°C water bath for 10 min, centrifuge at 60 g for 2 min and discard the supernatant; add digestion solution II (components: 0.25% trypsin, 0.5 mg / ml DNaseI), digest in a 37°C water bath for 10 min, and add an equal volume of fetal bovine serum to terminate the digestion;

[0062] (3) The digested primary cells (denoted as P1) were inoculated into a 6 cm culture dish and cultured with conventional DMEM high-glucose medium until the cell confluence was 80%. The cells were digested and passaged using 0.25% trypsin (denoted as P2). The P2 passage giant panda kidney cells were inoculated into a 24-well plate (inoculation density was 2 × 10 4 cells / well, and then cultured with modified DMEM high-glucose medium (DMEM high-glucose, 10% FBS, 300 UI / mL penicillin, 260 μg / mL streptomycin, 0.8 μg / mL amphotericin, 20 ng / mL EGF (human), 20 μM resveratrol, 80 mM trehalose) for 72 h to obtain P3 generation giant panda primary kidney cells;

[0063] (4) The P3 generation giant panda primary kidney cells were infected with lentivirus carrying the neomycin resistance gene and the SV40-LT gene for 2 h at an MOI of 5. The cells were then passaged at 2×10 4 The cells were seeded in 48-well plates at a concentration of 10 cells / mL, and 3 replicates were seeded in each way. After 48 h of culture, the cells were treated with screening medium (DMEM high glucose, 10% FBS, 15 ng / mL EGF, 15 ng / mL hydrocortisone, 15 μg / mL insulin, 12 μM resveratrol, 80 mM trehalose, and 1000 μg / mL G418) for 24 h to obtain the giant panda immortalized kidney cell line GPK-1.

[0064] Comparative Example 1

[0065] Compared with Example 1, the difference is that in step (2), only 0.25% trypsin is used for digestion, and digestion is carried out in a 37°C water bath for 10 min. This method is the currently reported conventional digestion method for kidney cells.

[0066] Then, the cell viability after the process in Comparative Example 1 and the process in Example 1 was detected respectively, and the results are shown in Table 1.

[0067] Table 1 Cell viability

[0068]

[0069] The results show that the method of step-by-step digestion with multiple digestive enzymes provided by the present invention can greatly improve the viability of the obtained cells, and the improvement effect is more obvious for field samples with lower quality. (Three replicates are set for each digestion method, * indicates that the cell viability obtained by the two digestion methods is significantly different, *P<0.05)

[0070] Comparative Example 2

[0071] Compared with Example 1, the difference is that 50 mL PBS solution is used as the protective solution in step (1).

[0072] Comparative Example 3

[0073] Compared with Example 1, the difference is that in step (1), 10 mL of PBS solution containing 50 μM resveratrol is used as the protective solution.

[0074] Comparative Example 4

[0075] Compared with Example 1, the difference is that in step (1), 10 mL of PBS solution containing 30 μM melatonin is used as the protective solution.

[0076] Comparative Example 5

[0077] Compared with Example 1, the difference is that in step (1), 10 mL of PBS solution containing 50 μg / ml giant panda milk cell exosome extract is used as the protective solution.

[0078] The cell viability after digestion treatment according to the schemes described in Example 1 and Comparative Examples 2 to 5 was tested respectively, and the results are shown in Table 2.

[0079] Table 2 Cell viability

[0080]

[0081] The results show that the tissue separation protection solution provided by the present invention can greatly improve the viability of the obtained cells, and the improvement effect is more obvious for field samples with lower quality. (Three replicates were set for each digestion method, * indicates that the obtained cell viability is significantly different, *P<0.05)

[0082] Comparative Example 6

[0083] Compared with Example 1, the difference is that in step (3), the modified DMEM high-glucose medium is not used for culturing.

[0084] Comparative Example 7

[0085] Compared with Example 1, the difference is that the modified DMEM high-glucose culture medium in step (3) includes: DMEM high-glucose, 10% FBS, 300 UI / mL penicillin, 260 μg / mL streptomycin, 0.8 μg / mL amphotericin, and 20 ng / mL EGF (human source).

[0086] Comparative Example 8

[0087] Compared with Example 1, the difference is that the modified DMEM high glucose medium in step (3) includes: DMEM high glucose, 10% FBS, 300 UI / mL penicillin, 260 μg / mL streptomycin, 0.8 μg / mL amphotericin, 20 ng / mL EGF (human source), and 10 μM resveratrol.

[0088] The proliferation multiples of the P3 generation cells obtained by the schemes described in Example 1 and Comparative Examples 6 to 8 were detected respectively, as follows:

[0089] After 72 hours of cell culture, all cells were digested and counted. Three replicates were set for each method. Since the initial number of cells was the same, the counting result after 72 hours could reflect the proliferation of cells. The cell proliferation multiple after 72 hours = the number of cells after 72 hours of culture / the initial number of cells inoculated (i.e., 2×10 4 The results are shown in Table 3.

[0090] Table 3 Cell proliferation multiples

[0091]

[0092] Note: The values ​​are expressed as mean ± standard deviation. * The results of single factor variance analysis show that the effect of the present invention is significantly higher than that of comparative examples 6 to 8 ( P <0.05).

[0093] According to the test results in Table 3, the present invention found that the improved DMEM high-glucose culture medium constructed by adding EGF, resveratrol and trehalose can greatly improve the proliferation effect of giant panda kidney primary cells.

[0094] Experimental Example 1: Study on the Establishment of Immortalized Cell Lines from Giant Panda Kidney Cells

[0095] By comparing conventional kidney cell isolation and culture with the tissue isolation and culture technology system used in the present invention, it was found that the low quality of primary cells was the key factor leading to the failure of previous successful cell establishment. Kidney tissues of captive 2-month-old individuals who died naturally were selected, and cells were obtained in the following 4 ways and cultured to P3 for virus infection.

[0096] Method 1: Activate tissues by the method of Comparative Example 2 (control), digest cells by the method of Comparative Example 1 (control), and culture cells by the method of Comparative Example 6 (control).

[0097] Method 2: Activate tissues in the manner of Example 1 (optimal), digest and obtain cells in the manner of Comparative Example 1 (control), and culture cells in the manner of Comparative Example 6 (control).

[0098] Method 3: Activate tissues in the manner of Example 1 (optimal), digest and obtain cells in the manner of Example 1 (optimal), and culture cells in the manner of Comparative Example 6 (control).

[0099] Method 4: adopt the technical solution of Example 1.

[0100] The P3 giant panda primary kidney cells (from methods 1-4) were infected with lentivirus carrying the neomycin resistance gene and SV40-LT gene for 2 h at an MOI of 5. The cells were then passaged at 2×10 4 The cells were inoculated in 48-well plates at a concentration of 10 cells / well, and 3 replicates were inoculated in each method. After 48 hours of culture, the cells were treated with screening medium (components: DMEM high glucose, 10% FBS, 10 ng / mL EGF, 10 ng / mL hydrocortisone, 10 μg / mL insulin, 10 μM resveratrol, 50 mM trehalose, and 800 μg / mL G418) for 24 hours, and 3 wells without G418 were retained in each method 1-4 as controls. At this time, the dead cells of the floating cells were removed, the remaining cells were digested and counted, and the survival rate of the cells in methods 1-4 was calculated = (the number of remaining cells in the group with G418 added / the number of cells in the group without G418 added) × 100%. Successfully infected cells will show neomycin resistance, so the survival rate after screening reflects the effect of cell infection by the virus. The higher the survival rate after screening, the better the infection effect of the virus. The results are shown in Table 4.

[0101] Table 4 Cell survival rate

[0102]

[0103] The results in Table 4 show that the giant panda kidney digestion and culture technology system provided by the present invention can significantly improve cell quality, thereby improving the effect of viral infection. The giant panda immortalized kidney cell line GPK-1 (obtained by screening and culturing the cells in duplicate well 2) was successfully established using method 4 (Example 1 of the present invention).

[0104] Experimental Example 2 Identification and Research of Giant Panda Immortalized Kidney Cell Line GPK-1

[0105] To further verify the immortalization effect of GPK-1, the following experiments and tests were performed.

[0106] 1. GPK-1 cell morphology assessment

[0107] The giant panda immortalized kidney cell line GPK-1 was observed by microscopy to have differences in cell morphology from primary cells at P10, P30 and P50. Figure 1 .

[0108] like Figure 1 As shown, the giant panda immortalized kidney cell line GPK-1 obtained by the present invention can be continuously cultured in vitro for more than 50 generations. Microscopic observation showed that GPK-1 had a cell morphology similar to that of primary cells at P10, P30 and P50, indicating that the giant panda immortalized kidney cell line GPK-1 obtained by the present invention can still maintain the original cell morphology after long-term culture.

[0109] 2. GPK-1 growth status assessment

[0110] The 3rd generation of kidney cells, the 30th generation of GPK-1 cells, and the 50th generation of GPK-1 cells were inoculated into 48-well plates with a starting inoculum of 4000 / mL per well. The cells were digested every 24 hours and counted using a cell counter to determine the cell concentration. After 9 consecutive days of counting, a cell growth curve was drawn with the culture time as the horizontal axis and the cell density as the vertical axis. The results are shown in Figure 2 .

[0111] like Figure 2 As shown, the cell proliferation ability of the giant panda immortalized kidney cell line GPK-1 obtained by the present invention is significantly stronger than that of the giant panda primary kidney cells. The growth curves of GPK-1-P30 and GPK-1-P50 are both "S"-shaped. During the 1-4 days of culture, the cells grow slowly and the cell number does not change much; after 4 days of culture, the cells begin to grow rapidly; after 8-9 days of culture, the cell growth rate slows down.

[0112] 3. GPK-1 cell type detection

[0113] The P3 generation giant panda primary kidney cells and the P50 generation GPK-1 were inoculated into 24-well plates and immunocytochemical staining was performed. Figure 3 .

[0114] like Figure 3 As shown, the results showed that primary renal cells ( Figure 3 A) CK14, CK18, and Vimentin are all expressed, but CK14 is expressed at a low level. Some cells do not express Vimentin, indicating mixed cells; GPK-1 cell line ( Figure 3 B) CK14, CK18, and Vimentin were all expressed, with weak fluorescence signal and low expression of CK14 and stable expression of CK18, indicating that the purity of GPK-1 was improved after transfection and screening, and it has epithelial cell characteristics. The results of RT-PCR further confirmed this conclusion ( Figure 3 C), and also showed that GPK-1 cells were successfully introduced with the SV40 gene by lentiviral transfection.

[0115] 4. Karyotype identification of GPK-1 cells

[0116] The karyotype of GPK-1 cells at generation P50 was identified, such as Figure 4 As shown, the cells with normal karyotype in the giant panda immortalized kidney cell line GPK-1 at generation P50 have 42 chromosomes, which is the same as the chromosome number of the giant panda.

[0117] Experimental Example 3 Application Research of Giant Panda Kidney-derived Cells GPK-1

[0118] The present invention further tests the feasibility of using the obtained giant panda immortalized kidney cell line GPK-1 in giant panda parvovirus infection experiments, and the specific process is as follows:

[0119] The giant panda kidney cell line GPK-1 cells were infected with FPV, and immunofluorescence experiments were performed 48 hours after infection and observed under a fluorescence microscope ( Figure 5 ). Red fluorescence indicates cells, and green fluorescence indicates FPV protein, indicating that the parvovirus has successfully and efficiently infected GPK-1 cells. This study not only confirms that the GPK-1 cell line can be used as a tool to evaluate the infection ability of parvovirus, but also has good potential as a model for cytopathology and toxicology research.

[0120] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a giant panda immortalized kidney cell line, characterized in that: The following steps are involved: (1) Collect kidney tissue from a dead giant panda and soak it in a protective solution for 10 to 20 minutes; the protective solution includes 50 to 80 μM resveratrol, 30 to 50 μM melatonin, and 50 to 100 μg / mL giant panda milk cell exosome extract; the preparation method of the giant panda milk cell exosome extract is as follows: Giant panda milk cells cultured for 1-5 generations were taken. When the cell confluence reached 80%, the complete medium was discarded, the adherent cells were washed, low-glucose DMEM without FBS was added and cultured for 48 hours, and the cell supernatant was collected and centrifuged to obtain giant panda milk cell exosome extract; (2) performing two-step digestion on the kidney tissue treated in step (1); the specific process of the two-step digestion is as follows: Digest in digestion solution I at 37°C for 10-15 min. After centrifugation, continue digestion in digestion solution II for another 10-15 min. The digestion solution I includes DMEM / F12, type IV collagenase at a concentration of 2 to 5 mg / mL, hyaluronidase at a concentration of 1 to 5 mg / mL, and DNaseI at a concentration of 0.1 to 0.5 mg / mL; the digestion solution II includes DNaseI at a concentration of 0.1 to 0.5 mg / mL, and 0.25% trypsin; (3) The digested cells are cultured and subcultured, and then the subcultured cells are infected with a lentivirus carrying the neomycin resistance gene and the SV40-LT gene, and then the giant panda immortalized kidney cell line is screened and established.

2. The preparation method according to claim 1, characterized in that: The process of culturing and subculturing the digested cells in step (3) is as follows: The cells digested in step (2) were inoculated into a culture dish, cultured in DMEM high-glucose medium until the cell confluence was 80%, and after digestion and passage, cultured in DMEM high-glucose modified medium for 72 to 96 hours; The DMEM high-glucose modified culture medium comprises DMEM high-glucose, 10% FBS, 200-300 UI / mL penicillin, 200-260 μg / mL streptomycin, 0.5-1.2 μg / mL amphotericin, 10-20 ng / mL EGF, 10-20 μM resveratrol and 50-80 mM trehalose.

3. The preparation method according to claim 1, characterized in that: The culture medium used for screening in step (3) includes DMEM high glucose, 10% FBS, 10-15 ng / mL EGF, 10-15 ng / mL hydrocortisone, 10-15 μg / mL insulin, 10-12 μM resveratrol, 50-80 mM trehalose, and 800-1000 μg / mL G418.

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