Construction method and application of sika deer immortalized renal epithelial cell line

Abstract

The invention relates to the technical field of cell engineering, and particularly discloses a construction method and application of a sika deer immortalized renal epithelial cell line, and the construction method comprises the following steps: taking renal epithelial primary cells from healthy sika deer renal cortex tissues to obtain primary renal epithelial cells; treating the primary renal epithelial cells with trypsin to obtain a cell suspension; inoculating the cell suspension into a complete culture medium, and transfecting with lentivirus loaded with SV40 large T antigen genes to obtain transfected cells; 3 [mu] g / mL puromycin is applied to the transfected cells for selective culture, so that non-transfected cells are eliminated, and drug-resistant cells are obtained; carrying out continuous passage on the drug-resistant cells for at least 30 generations to obtain the immortalized renal epithelial cell line of the sika deer; according to the invention, through a specific action mechanism (inhibiting a p53 / pRb pathway and blocking cell cycle exit) of the SV40 large T antigen, a proliferation limit caused by inhibition of telomerase activity of primary cells of the cervidae animals is overcome, and a cell resource library capable of realizing continuous passage is established.

Description

Technical Field

[0001] The invention belongs to the technical field of cell engineering, and particularly relates to a construction method of an immortalized sika deer kidney epithelial cell line and an application thereof. Background Art

[0002] The kidney is a key organ in mammals for excreting metabolic waste, regulating water and salt balance, and maintaining a stable internal environment. Its functions are closely linked to overall health and play a central role in systemic physiological regulation. It holds a crucial position in physiological and medical research, making in vitro studies of renal cells and their functions indispensable. The nephron, composed of glomeruli and renal tubules, possesses a complex and highly organized structure, with a rich vascular network. Renal tissue contains a variety of cells, with renal epithelial cells and podocytes being the primary targets of research. These cells play crucial roles in filtration, reabsorption, and secretion, and are closely associated with the development and progression of kidney disease.

[0003] The isolation and culture of renal epithelial cells in vitro are of great research value. Currently, researchers have successfully isolated and cultured renal epithelial cells from a variety of animals, including humans, mice, rabbits, cows, dogs, and cats. Commonly used methods include enzymatic digestion, tissue explant culture, and enzymatic filtration. Primary renal epithelial cells cultured in vitro have the advantage of controlling a single variable and can effectively eliminate the interference of other factors. However, their limitation is that the number of passages is limited, which not only restricts the in vitro study of kidney disease, but also requires repeated acquisition of primary cells, consuming a lot of manpower, material resources, and financial resources. In contrast, immortalized cells not only overcome these shortcomings, but also have advantages that primary cells lack, such as good cell uniformity, strong stability, and simple culture. Therefore, compared with primary renal epithelial cells, obtaining immortalized renal epithelial cells has important practical significance.

[0004] SV40 is an enveloped, double-stranded, circular DNA virus discovered and isolated in the early 1960s. It belongs to the Polyomaviridae family and can infect monkey cells and induce tumors. Studies have shown that the SV40 gene fragment is one of the most commonly used target fragments for inducing cell immortalization. After the SV40 gene is transferred into cells, the SV40-encoded T protein binds to the tumor suppressor protein (p53) and the retinoblastoma protein (pRb), preventing the normal expression of tumor suppressor genes. It also mediates the transformation of normal cells by preventing the transition between the G1 / S and G2 / M phases of the cell cycle, thereby inhibiting apoptosis, prolonging the cell cycle, and allowing cells to proliferate indefinitely.

[0005] The SV40 gene has been widely used to immortalize a variety of cells, including human pericyte lines and human dermal fibroblasts, providing a valuable tool for exploring malignant tumor progression and viral-genomic interactions. Compared to immortalization methods involving ectopic expression of telomerase or reverse transcriptase, SV40 antigen gene-mediated immortalization offers advantages such as high infection efficiency, wide applicability, and improved safety. However, a disadvantage is that the SV40 antigen gene may alter gene expression in immortalized cells. While ectopic expression of telomerase or remote reverse transcriptase can introduce telomerase into cells, it can help normal cells transition through senescence and the critical phase without causing cancerous transformation. However, for some cells, restoring telomerase activity is insufficient for immortalization and requires the combined introduction of other genes. Furthermore, immortalization methods involving oncogenes or radiation induction are not only inefficient but also carry a high risk of tumorigenicity. In contrast, SV40 antigen gene-mediated immortalization systems are more stable and their tumorigenicity is relatively controllable.

[0006] Although cell culture technology is relatively mature, due to differences in species and cell types, there are still significant differences in the culture methods and growth characteristics of primary cells from different sources. At present, renal epithelial cell lines have been successfully established in many animals, but there have been no reports on the use of SV40 virus to induce the immortalization of sika deer renal epithelial cells. Although primary cell culture is an important research model, due to the inhibition of its telomerase activity, the telomeres gradually shorten during continuous passage, resulting in limited cell proliferation capacity. In addition, primary culture also has problems such as cumbersome operation, long experimental cycle, and susceptibility to contamination, all of which limit its widespread application. Therefore, the establishment of a stably passaged in vitro culture model of sika deer renal epithelial cells is of great significance for promoting the establishment of sika deer-related disease models, drug screening, gene function research, and the development of biological products.

[0007] To solve the above problems, the present application proposes a method for constructing an immortalized sika deer renal epithelial cell line and its application. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for constructing an immortalized sika deer renal epithelial cell line and its application, so as to solve the problems raised in the above background technology.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A method for constructing an immortalized sika deer renal epithelial cell line comprises the following steps:

[0011] Primary renal epithelial cells were obtained from the renal cortex tissue of healthy sika deer to obtain primary renal epithelial cells;

[0012] The primary renal epithelial cells were digested with a reagent containing 0.25% trypsin to obtain a cell suspension;

[0013] The cell suspension is inoculated into complete culture medium and cultured and passaged to obtain a cell population ready for transfection;

[0014] transfecting the cell population with a lentivirus carrying the SV40 large T antigen gene to obtain transfected cells;

[0015] Applying 3 μg / mL puromycin to the transfected cells for selection and culture to eliminate untransfected cells and obtain drug-resistant cells;

[0016] The drug-resistant cells were continuously passaged for at least 30 generations to obtain an immortalized sika deer kidney epithelial cell line.

[0017] Preferably, the primary renal epithelial cells are digested with a reagent containing 0.25% trypsin to obtain a cell suspension, comprising:

[0018] The renal cortex tissue was obtained from the kidney tissue of a healthy 26-day-old male sika deer. The kidney tissue was washed in PBS containing 2 times penicillin-streptomycin, the connective tissue was removed and disinfected with 75% alcohol for 15 to 30 seconds, and then repeatedly rinsed with PBS. The renal cortex part was cut and minced before enzymatic digestion; the enzymatic digestion used 0.25% (w / v) trypsin, oscillated in a 37°C water bath for 10 minutes, repeated 4 to 6 times, and the supernatant was collected and filtered through a 100-mesh sieve, centrifuged at 300×g for 5 minutes, and the supernatant was discarded and resuspended in epithelial cell complete culture medium, and differentially adhered for 1 hour to obtain purified primary renal epithelial cells.

[0019] Preferably, the trypsin digestion treatment conditions for primary renal epithelial cells are as follows: at 37° C. and 5% CO 2 , 0.25% (w / v) trypsin is used for 10 to 15 minutes, and cells are gently pipetted every 5 minutes (using a 1 mL pipette tip to pipet 3 times) to promote cell detachment; after digestion is terminated, an equal volume of DMEM F12 medium containing 10% fetal bovine serum is added for neutralization, and the mixture is centrifuged at 300×g for 5 minutes, and the supernatant is discarded to obtain a cell suspension;

[0020] The DMEM F12 culture medium also includes:

[0021] 10% (v / v) fetal bovine serum, 1% (v / v) penicillin-streptomycin (100 U / mL; 100 μg / mL), 5 μg / mL epidermal growth factor (EGF), 2 mM L-glutamine and 1 mM sodium pyruvate; the cell seeding density was 1.0×10 5 cells / cm 2After culturing at 37°C and 5% CO2 for 48 h, the cells were passaged with 0.25% trypsin at a passage ratio of 1:3.

[0022] Preferably, the step of transfecting the cell population B with a lentivirus carrying the SV40 large T antigen gene to obtain transfected cells comprises the following steps:

[0023] S41. Clone the optimized human SV40 large T antigen gene (including nuclear localization signal) into the pLV-EF1α-Puro vector;

[0024] S42, co-transfected into HEK293T cells with pLP1:pLP2:pLP / VSVG:pLV-SV40 LT at a mass ratio of 4:3:1:5;

[0025] S43, 24 h after transfection, the medium was replaced, and the supernatant was collected at 48 h and 72 h, filtered through 0.45 μm, concentrated with 8% PEG8000 at 4 °C for 12 h, and centrifuged at 4000 × g for 30 min;

[0026] S44. Adjust the concentrated virus to MOI = 10-20 (optimized according to the survival rate of the third generation cells), add 8 μg / mL polybrene to enhance infection, and replace the culture medium containing virus inactivator after incubation for 12 hours.

[0027] Preferably, the lentiviral vector of the SV40 large T antigen gene used for transfection is constructed with EF1α-SV40-IRES-puromycin and carries a resistance gene; about 1×10 5 After 24 hours of adherence, 1 mL of complete culture medium and 20 μL of viral supernatant were added to the cells for infection at an MOI of about 10 to 20 for 12 hours. Fresh culture medium was then replaced and the cells were subcultured into T25 flasks for expansion.

[0028] Preferably, 0.05 million untransfected primary cells are plated in a 24-well plate and treated with 1-7 μg / mL puromycin. The culture medium is changed every 2 days. The lowest concentration that can kill all control cells within 4 days is 3 μg / mL, and this concentration is continuously screened for 4 days to obtain drug-resistant cells.

[0029] Preferably, the drug-resistant cells are serially passaged for 30 to 60 generations, and the following characteristics are performed at the 10th, 35th, and 60th generations, respectively:

[0030] Morphology: Under phase contrast microscopy, cells are arranged in a typical cobblestone-like pattern;

[0031] Karyotype: Giemsa staining detected 2n = 66 normal chromosomes;

[0032] SV40 LT gene expression: RT-qPCR Ct value <25, Western blot relative expression level was significantly higher than that of primary cells;

[0033] Growth curve: measured by CCK-8 method, the cells entered the logarithmic growth phase on the 3rd to 4th day, reached the peak on the 5th day, and cell death was observed on the 6th day;

[0034] Apoptosis rate: <5% detected by Annexin V / PI double staining flow cytometry.

[0035] Preferably, SV40 LT gene expression is detected by RT-PCR: total RNA is extracted from cells and the expression is detected by PrimeScript TM Reverse transcription and PCR amplification were performed using the FAST RT kit. The SV40 LT primer sequences used were F: ttc aga gca gaa ttg tggagt g, and R: cct ggc gtg ctt cat cat cat c. The amplified product was only detected in immortalized cells of passage 30 and above but not in primary cells.

[0036] A method for constructing an immortalized sika deer renal epithelial cell line and its application in establishing an in vitro model of sika deer kidney disease.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The present invention achieves the stable immortalization of sika deer renal epithelial cells for the first time, filling the gap in the kidney research model of this species. Through the specific mechanism of action of SV40 large T antigen (inhibiting the p53 / pRb pathway and blocking cell cycle exit), the proliferation limit caused by the inhibition of telomerase activity in primary cells of deer animals is overcome, and a cell resource library with sustainable passage is established. The immortalized cell line has long-term genetic stability and functional consistency, avoiding experimental deviations caused by individual differences in primary cells. Compared with the tedious process of repeatedly isolating primary cells, this cell line provides a standardized research platform, significantly shortening the experimental cycle and reducing research costs.

[0039] (2) As an in vitro simulation system of the kidney unique to sika deer, the present invention can accurately reflect the metabolic characteristics of deer-derived drugs and the nephrotoxicity response. Its enhanced migration ability and maintained epithelial barrier function provide an ideal model for the study of renal tubular injury repair mechanisms and the screening of anti-fibrotic drugs. The anchorage-independent growth characteristics of immortalized cells were confirmed by soft agar cloning experiments, and combined with karyotype stability verification, the tumorigenic risk was eliminated. SV40-mediated immortalization did not induce malignant transformation, ensuring its feasibility as a safe carrier in the production of biological products.

[0040] (3) The present invention provides a reproducible cellular-level research tool for studying the mechanism of sika deer kidney diseases (such as chronic wasting disease and glomerulonephritis), and supports applications such as expression of deer-derived functional proteins and development of viral vaccines, thereby promoting cross-innovation between the characteristic animal husbandry industry and biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a morphological diagram of primary renal epithelial cells of sika deer under a microscope of the present invention;

[0042] Figure 2 This is the immunofluorescence identification result diagram of the present invention;

[0043] Figure 3 This is the result of transfection for 12 h and screening for 4 days of the present invention;

[0044] Figure 4 The transfected sika deer kidney epithelial cells and primary cells of the present invention;

[0045] Figure 5 This is a diagram of the cell migration structure of the present invention;

[0046] Figure 6 This is a graph showing the growth curve of renal epithelial cells analyzed by CCK-8 of the present invention;

[0047] Figure 7 This is a split phase photographic analysis diagram of the present invention;

[0048] Figure 8 This is the SV40-TL gene map detected by RT-PCR of the present invention;

[0049] Figure 9 This is a graph showing the CK18 expression in immortalized renal epithelial cells detected by IFA of the present invention;

[0050] Figure 10 It is a cell soft agar clone diagram of the present invention. DETAILED DESCRIPTION

[0051] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0052] Example 1:

[0053] See also Figures 1 to 10 As shown, a method for constructing an immortalized sika deer renal epithelial cell line comprises the following steps:

[0054] 1. Isolation and purification of primary sika deer kidney epithelial cells:

[0055] 1. Sample source: A healthy 26-day-old male sika deer was selected from a farm in Changchun City, Jilin Province. It was quarantined before sampling and met the experimental requirements.

[0056] 2. After sacrificing the sika deer, the abdominal cavity was opened and the kidneys isolated. The tissue was placed in PBS containing 2x P / S, placed on ice, and brought back to the laboratory for cell isolation within 2 hours. The kidneys were washed in PBS containing double-antibody (100 U / ml penicillin and 100 U / ml streptomycin), excess connective tissue removed, and placed in 75% alcohol for 15-30 seconds. They were then immediately washed in fresh PBS buffer. Renal epithelial cells were isolated and cultured using trypsin digestion. Sika deer kidney samples were taken, and the renal cortex of the kidney was cut with surgical scissors. The cells were repeatedly rinsed with PBS until the liquid was clear. The collected renal cortex was minced and washed with PBS. The supernatant was discarded. At least 3 volumes of 0.25% trypsin were added to the precipitate, and the cells were digested in a 37°C water bath with shaking for 10 minutes. The supernatant was aspirated, FBS was added to terminate the digestion, and trypsin was added to the precipitate again to continue digestion. This was repeated 4-6 times, and the supernatant was collected. The collected digestion solution was passed through a 100-mesh sieve, the filtrate was collected, and the cells were centrifuged at 300×g for 5 minutes. The supernatant was discarded, and the precipitate was resuspended in epithelial complete medium (containing 1% P / S, 1% amphotericin B, and 1% gentamicin). The precipitate was plated and placed in a 37°C, 5% CO2 cell culture incubator for differential attachment: After 1 hour, the supernatant of the non-adherent cells was extracted, and the cells were cultured in a new culture flask to preliminarily obtain primary sika deer renal epithelial cells. Observe the cells under a microscope and culture for about 72 hours. Most cells have clear outlines, typical paving stone shape, epithelial cell morphology, uniform cell density distribution, and single-layer growth. Figure 1 shown.

[0057] 2. Immunofluorescence detection of CK18 expression in cells:

[0058] Primary sika deer kidney epithelial cells were seeded into a 48-well plate. 1 mL of culture medium was added to each well, along with 0.02 million cells per well. The cells were incubated for 2 hours or overnight. Staining was performed when the cells reached 50-60% confluency. The procedure was as follows:

[0059] (1) Discard the cell culture medium, carefully wash with PBS three times, and then add 200 μL of 4% paraformaldehyde to fix the cells at 4°C for 30 minutes.

[0060] (2) Discard the paraformaldehyde, carefully wash with PBS three times, and use membrane blocking solution (0.5% Triton X-100 mixed with PBS in a ratio of 1:1, and add 10% goat serum) to block the cell membrane for 2 hours.

[0061] (3) Wash with PBS three times, dilute CK18 antibody and Vimentin in a ratio of 1:200 using primary antibody diluent, add to the culture wells, and incubate at 4°C overnight.

[0062] (5) Wash with PBS three times, add secondary antibody, incubate at room temperature for 2 h, and rinse with PBS three times, 5 min each time.

[0063] (6) Remove the PBS solution and add 100 μL of freshly prepared DAPI working solution. Immediately observe under an inverted microscope and take photos.

[0064] CK18 is a specific marker protein for epithelial cells. Immunofluorescence identification revealed that all isolated primary sika deer kidney epithelial cells expressed CK18. The cytoplasm of positive cells was stained green and the nucleus was blue, but they did not express the fibroblast marker protein Vimentin, confirming that there was no fibroblast contamination in the cells. Figure 2 Immunofluorescence identification results:

[0065] 3. Transfection:

[0066] 3.1 Basic information of SV40 overexpression lentivirus is shown in Table 1 below:

[0067] Table 1

[0068]

[0069] 3.2 Transfection:

[0070] The cells were seeded into 6-well plates, with the number of cells per well being approximately 1 × 10 5 After culturing for 24 h until the cells adhered to the wall, the medium was changed, 1 mL of complete medium was added, and 20 μL of SV40 overexpression lentivirus was added. The culture was continued after mixing. The cell status was observed after 12 h, and the medium was replaced with fresh medium. When the cells were confluent, they were subcultured into T25 culture flasks for expansion culture.

[0071] 3.3 Determination of the kill curve:

[0072] Untransfected primary renal epithelial cells were plated into 24-well plates at 0.05 million per well. After culturing overnight for 24 hours, the old culture medium in the 24-well plates was removed and fresh culture medium containing different concentrations of puromycin (1ug / mL, 2ug / mL, 3ug / mL, 4ug / mL, 5ug / mL, 6ug / mL, 7ug / mL) was added to the 24-well plates containing cells. Fresh screening culture medium was replaced every 2 days, and the survival rate of cells was observed every day. The minimum puromycin concentration used was the lowest screening concentration that killed all cells within 1-4 days from the start of puromycin screening. The experimental results showed that the concentration of puromycin used was 3μg / mL and the action time was 4 days.

[0073] 3.4 Puromycin selection of transfected cells:

[0074] Untransfected primary renal epithelial cells were plated into 24-well plates at 0.05 million per well and incubated overnight. After 24 hours of overnight culture, the old culture medium in the 24-well plates was removed and a selection medium containing 3 μg / mL puromycin was added and incubated. Fresh selection medium was replaced every 2 days. The survival rate of cells was observed every day. Cells that survived at the same time point (4 days) were considered to be successfully transfected cells, and the selected cells were amplified.

[0075] The screened cells were expanded and the cells after screening were P1. The immortalized deer kidney epithelial cell line was successfully established when the cells were expanded to at least 30 generations. In our laboratory, the cells have been subcultured to 60 generations. Figure 3 The results are shown after 12 hours of transfection and 4 days of screening.

[0076] 4. Analysis of cell growth characteristics:

[0077] 4.1 Cell morphology analysis:

[0078] After transfection, the morphological changes of the cells were observed under a phase contrast microscope and compared with the primary cells. The second generation of primary renal epithelial cells and the renal epithelial cells cultured to the 35th generation after transfection were inoculated into 24-well plates. When the cell confluence reached 30%, they were fixed with 4% paraformaldehyde, stained with Giemsa, observed under a microscope, and photographed. The results showed that the transfected sika deer renal epithelial cells were similar in morphology to the primary cells, with obvious central nuclei and clearly visible nucleoli. Most cells were polygonal or oval in shape, and the cells were arranged in a typical cobblestone-like pattern. There was contact inhibition between the cells. The results are as follows: Figure 4 As shown;

[0079] 4.2 Cell Migration

[0080] Collect 2×10 4 Second-generation primary renal epithelial cells and 55th-generation renal epithelial cells after transfection were resuspended in 200 μL serum-free DMEM / F12 medium and inoculated into Transwell l cell chambers (pore size 8 μm). 500 μL DMEM / F12 complete medium was added to the lower chamber; the cells were cultured in a 37°C, 5% CO2 incubator for 24 h; the cells that passed through the upper chamber membrane were fixed with 4% paraformaldehyde and stained with crystal violet, and the cells that did not migrate on the surface of the upper chamber membrane were wiped off with a cotton swab, observed under a microscope, and photographed.

[0081] Transwel l results showed that both primary cells and transfected cells could pass through the Transwel l chamber and had migration ability, and the migration ability of immortalized cells was better than that of primary cells. Figure 5 As shown;

[0082] 4.3 CCK-8 analysis of renal epithelial cell growth curve:

[0083] Primary renal epithelial cells and renal epithelial cells at passages 15 and 30 after transfection were seeded in 96-well plates at a density of 2,000 cells per well. 10 μL of CCK-8 was added to each well every 24 hours and incubated in a 37°C incubator in the dark for 2 hours. The OD value of each well was measured at a wavelength of 450 nm. The data were read three times each time and the average was taken. The measurement was continued for 7 days, and a growth curve was drawn with time as the horizontal axis and OD value as the vertical axis. The results are shown in Figure 2. Figure 6 As shown, renal epithelial cells proliferated slowly on the 1st and 2nd days after transfection, and then began to proliferate rapidly on the 3rd and 4th days, entering the logarithmic growth phase. The cells reached their peak on the 5th day, and began to die on the 6th day, with a significant decrease in proliferation capacity.

[0084] 5. Genetic characteristics analysis:

[0085] 5.1 Karyotype analysis

[0086] 1. Colchicine Treatment of Cells: Observe immortalized renal epithelial cells at passage 60 under a microscope. When the confluence of the cultured cells is approximately 70-80%, add 0.2 μg / mL colchicine to the culture medium and incubate in a 37°C, 5% CO2 incubator for 3 hours. Digest and collect the cells into a 15 mL centrifuge tube and centrifuge at 800 rpm for 5 minutes. Remove the supernatant and retain the cell pellet.

[0087] 2. Hypotonicity: Add 8 mL of 0.075 mol / L KCl hypotonic solution, preheated in a 37°C water bath. Mix thoroughly by pipetting. Incubate in a 37°C water bath for 15 minutes. Centrifuge at 800 rpm for 5 minutes, then discard the supernatant, leaving approximately 0.5 mL of residual solution to prevent cell loss.

[0088] 3. Pre-fixation: Gently scrape the bottom of the centrifuge tube and gently shake to resuspend the swollen cell pellet. Add freshly prepared fixative (methanol: glacial acetic acid = 3:1) dropwise along the tube wall, gently shaking the tube to mix thoroughly. Add approximately 1 mL of fixative and pre-fix for 5 minutes. Centrifuge at 800 rpm for 10 minutes, aspirate and discard the supernatant, leaving approximately 0.5 mL of residual liquid.

[0089] 4. First fixation: Gently vortex to resuspend the cells at the bottom, add 5 mL of fixative, gently pipette the cells several times, and incubate at room temperature in the dark for 30 minutes. Centrifuge at 800 rpm for 10 minutes, and discard the supernatant, leaving a residual volume of approximately 0.5 mL.

[0090] 5. Second fixation: Repeat step 4;

[0091] 6. Preparation: Wipe the slide with alcohol and let it dry. Avoid contact with your fingers to prevent fingerprints on non-frosted areas. Pipette a small amount of cell suspension and gradually drip it onto the slide from a height of 40-50 cm, avoiding any areas that have already been dripped. After dripping, immediately fix the slide several times with a quick flame of an alcohol lamp. The fixative evaporates, and distinct cell particles will appear on the slide.

[0092] 7G band staining: Place the baked slides in Giemsa stain for 10 minutes. After observing the mitotic phase under a microscope and seeing that it is purple-red, rinse the excess Giemsa stain on the slides with running water and take photos of the mitotic phase for analysis.

[0093] The results are as follows Figure 7 As shown, the number of chromosomes in the immortalized renal epithelial cells was still 66 when they were passed to the 60th generation, without loss or doubling, indicating that the genetic structure of the immortalized renal epithelial cells remained stable during unlimited passaging.

[0094] 5.2 RT-PCR detection of SV40-TL gene:

[0095] (1) Extraction of total cellular RNA:

[0096] Follow the instructions for the TransZol Up Plus RNA Kit. First, pour off the culture medium and rinse once with 1x PBS. Add 1 ml of TransZol Up per 10 cm of cultured cells. Place the tube horizontally for a moment to allow the lysis solution to evenly distribute on the cell surface and lyse the cells. Then, use a pipette to detach the cells (for firmly adhered cells, a cell scraper can be used to detach the cells). Transfer the cell lysate to a centrifuge tube, add 0.2 ml of RNA Extraction Agent, and repeatedly pipette until there is no visible precipitation in the lysate. Vortex and shake at room temperature for 5 minutes.

[0097] 2. Centrifuge at 10,000g for 15 minutes at 2-8°C. The sample will now separate into three layers: a colorless aqueous phase (upper layer), a middle layer, and a pink organic phase (lower layer). The RNA is in the aqueous phase, which should be approximately 50%-60% of the volume of the TransZolUp reagent used. (To avoid DNA contamination from the middle layer, retain a portion of the aqueous phase.)

[0098] 3. Transfer the colorless aqueous phase to a new centrifuge tube, add an equal volume of anhydrous ethanol (precipitate may appear at this time), and gently invert to mix. Subsequent centrifugation can be performed at room temperature.

[0099] 4. Add the resulting solution and precipitate to the centrifuge column, centrifuge at 12,000 x g for 30 seconds at room temperature, and discard the flow-through (if the volume is larger than the capacity of the centrifuge column, this can be done in several steps).

[0100] 5. Add 500 μl of CB9, centrifuge at 12000 g for 30 seconds at room temperature, and discard the flow-through.

[0101] 6. Repeat step 5 once.

[0102] 7. Add 500 μl WB9 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 x g for 30 seconds at room temperature, and discard the flow-through.

[0103] 8. Repeat step 7 once.

[0104] 9. Centrifuge at 12000g for 2 minutes at room temperature to completely remove residual ethanol.

[0105] 10. Place the spin column in the RNase-free Tube (provided in the kit), add 50-200 μl RNase-free water in the center of the spin column, and let it stand at room temperature for 1 minute.

[0106] 11. Centrifuge at 12000g for 1 min at room temperature to elute RNA.

[0107] 12. Store RNA at -80°C.

[0108] (2) Primer design:

[0109] The primer sequences used for RT-PCR of SV40 and GAPDH are shown in the table. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0110]

[0111] (3) cDNA synthesis according to PrimeScript TM Perform reverse transcription according to the instructions of the FAST RT reagent Kit with gDNA Eraser, using extracted RNA as a template. The steps are as follows:

[0112] Table 2 Genomic DNA removal reaction

[0113]

[0114] PCR reaction conditions: 42°C, 2 min; 4°C

[0115] Table 3 Reverse transcription reaction

[0116]

[0117] PCR reaction conditions: 37°C, 10 min; 85°C, 5 sec; 4°C

[0118] The reaction system is: 12.5 μL of TB Green Premix Ex Taq II, 1 μL of upstream primer and downstream primer, 8.5 μL of water, 1 μL of cDNA, and a total system of 25 μL. Place it in a qPCR instrument and set the program.

[0119] The results are as follows Figure 8 As shown, the SV40 antigen gene was expressed in immortalized renal epithelial cells at passage 30, but not in primary cells.

[0120] 5.3 IFA detection of CK18 expression in immortalized renal epithelial cells:

[0121] Immortalized renal epithelial cells at passage 50 were seeded into a 48-well plate. 1 mL of culture medium was added to each well, along with 0.02 million cells per well. The plate was incubated for 6 hours. Staining was performed when the cells reached 50-60% confluency. The procedure was as follows:

[0122] (1) Discard the cell culture medium, carefully wash with PBS three times, and then add 200 μL of 4% paraformaldehyde to fix the cells at 4°C for 30 minutes.

[0123] (2) Discard the paraformaldehyde, carefully wash with PBS three times, and use membrane blocking solution (0.5% Triton X-100 mixed with PBS in a ratio of 1:1, and add 10% goat serum) to block the cell membrane for 2 hours.

[0124] (3) Wash with PBS three times, dilute CK18 antibody and Vimentin in a ratio of 1:200 using primary antibody diluent, add to the culture wells, and incubate at 4°C overnight.

[0125] (5) Wash with PBS three times, add secondary antibody, incubate at room temperature for 2 h, and rinse with PBS three times, 5 min each time.

[0126] (6) Remove the PBS solution and add 100 μL of freshly prepared DAPI working solution. Immediately observe under an inverted microscope and take photos.

[0127] Immunofluorescence analysis revealed that all immortalized renal epithelial cells expressed CK18, with the cytoplasm of positive cells stained red and the nucleus blue, and no expression of the fibroblast marker protein Vimentin. Figure 9 The results are from immunofluorescence.

[0128] 6. Cell Soft Agar Cloning:

[0129] (1) Immortalized cells at passage 55 were taken and prepared into single-cell suspensions. Immortalized cells and primary cells at passage 2 were used as positive and negative cell controls, respectively. They were digested with 0.25% trypsin and gently pipetted to make single cells. Live cell counts were performed and the cell density was adjusted to 5 × 103 cells / mL using DMEM medium containing 20% ​​fetal bovine serum. Gradual dilutions were performed according to experimental requirements.

[0130] (2) Two low-melting-point agarose solutions with concentrations of 1.2% and 0.7% were prepared using purified Wahaha water. After high-temperature sterilization, they were kept at 40°C without solidification.

[0131] (3) Mix 1.2% agarose and 2x DMEM medium (containing 20% ​​newborn calf serum) in a 1:1 ratio, take 1.5 mL of the mixture and inject it into a 6-well plate. Cool and solidify it, which can be used as the bottom agar. Place it in a CO2 incubator for later use.

[0132] (4) After mixing 0.7% agarose and 2xDMEM culture medium in a sterile test tube at a ratio of 1:1, add 0.2 mL of cell suspension to the tube and mix thoroughly. Take 1.5 mL of the mixture and inject it into the plate covered with 1.2% agarose as the bottom layer to gradually form a double agar layer. After the upper layer of fat solidifies, place it in a 37°C, 5% CO2 incubator and culture for 21 days.

[0133] (5) Place the plate under an inverted microscope and observe whether colonies are formed.

[0134] The results are as follows Figure 10 As shown, no clonal cell clusters appeared in the primary cells and the 30th and 50th passages after transfection, indicating that the cells had no possibility of becoming cancerous.

[0135] As can be seen from the above, this invention achieves the first stable immortalization of sika deer renal epithelial cells, filling a gap in renal research models for this species. By leveraging the specific mechanism of action of the SV40 large T antigen (inhibiting the p53 / pRb pathway and blocking cell cycle exit), this method overcomes the proliferation limit caused by suppressed telomerase activity in primary deer cells, establishing a sustainable cell resource library.

[0136] Immortalized cell lines offer long-term genetic stability and functional consistency, eliminating experimental biases caused by individual differences in primary cells. Compared to the tedious process of repeatedly isolating primary cells, these cell lines provide a standardized research platform, significantly shortening experimental cycles and reducing research costs.

[0137] As a unique in vitro model of the sika deer kidney, the system accurately reflects the metabolic characteristics and renal toxicity responses of deer-derived drugs. Its enhanced migration capacity and maintained epithelial barrier function provide an ideal model for studying the mechanisms of renal tubular injury and repair and for screening anti-fibrotic drugs.

[0138] Soft agar cloning assays confirmed the anchorage-independent growth characteristics of the immortalized cells, while karyotypic stability was verified to eliminate the risk of tumorigenesis. SV40-mediated immortalization did not induce malignant transformation, ensuring its feasibility as a safe vector for biopharmaceutical production.

[0139] It provides replicable cell-level research tools for the study of the mechanisms of sika deer kidney diseases (such as chronic wasting disease and glomerulonephritis), supports applications such as deer-derived functional protein expression and viral vaccine development, and promotes cross-innovation between the characteristic livestock industry and biomedicine.

[0140] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing an immortalized sika deer renal epithelial cell line, characterized in that: The following steps are involved: Primary renal epithelial cells were obtained from the renal cortex tissue of healthy sika deer to obtain primary renal epithelial cells; The primary renal epithelial cells were digested with a reagent containing 0.25% trypsin to obtain a cell suspension; The cell suspension is inoculated into complete culture medium and cultured and passaged to obtain a cell population ready for transfection; transfecting the cell population with a lentivirus carrying the SV40 large T antigen gene to obtain transfected cells; Applying 3 μg / mL puromycin to the transfected cells for selection and culture to eliminate untransfected cells and obtain drug-resistant cells; The drug-resistant cells were continuously passaged for at least 30 generations to obtain an immortalized sika deer kidney epithelial cell line.

2. The method for constructing an immortalized sika deer renal epithelial cell line according to claim 1, characterized in that: The primary renal epithelial cells are digested with a reagent containing 0.25% trypsin to obtain a cell suspension, comprising: The renal cortex tissue was obtained from the kidney tissue of a healthy 26-day-old male sika deer. The kidney tissue was washed in PBS containing 2 times penicillin-streptomycin, the connective tissue was removed and disinfected with 75% alcohol for 15 to 30 seconds, and then repeatedly rinsed with PBS. The renal cortex part was cut and minced before enzymatic digestion; the enzymatic digestion used 0.25% (w / v) trypsin, oscillated in a 37°C water bath for 10 minutes, repeated 4 to 6 times, and the supernatant was collected and filtered through a 100-mesh sieve, centrifuged at 300×g for 5 minutes, and the supernatant was discarded and resuspended in epithelial cell complete culture medium, and differentially adhered for 1 hour to obtain purified primary renal epithelial cells.

3. The method for constructing a sika deer immortalized renal epithelial cell line according to claim 2, characterized in that: The trypsin digestion conditions for primary renal epithelial cells were as follows: 0.25% (w / v) trypsin was used for 10-15 minutes at 37°C and 5% CO2, and cells were gently pipetted every 5 minutes to promote cell detachment. After digestion, an equal volume of DMEM F12 medium containing 10% fetal bovine serum was added for neutralization, and the cells were centrifuged at 300×g for 5 minutes, and the supernatant was discarded to obtain a cell suspension. The DMEM F12 culture medium also includes: 10% fetal bovine serum, 1% penicillin-streptomycin, 5 μg / mL epidermal growth factor, 2 mM L-glutamine and 1 mM sodium pyruvate; the cell seeding density was 1.0×10 5 cel ls / cm 2 After culturing at 37°C and 5% CO2 for 48 h, the cells were passaged with 0.25% trypsin at a passage ratio of 1:

3.

4. The method for constructing a sika deer immortalized renal epithelial cell line according to claim 1, characterized in that: The cell population B is transfected with a lentivirus carrying the SV40 large T antigen gene to obtain transfected cells, comprising the following steps: S41. Clone the optimized human SV40 large T antigen gene into the pLV-EF1α-Puro vector; S42, co-transfected into HEK293T cells with pLP1:pLP2:pLP / VSVG:pLV-SV40 LT at a mass ratio of 4:3:1:5; S43, 24 h after transfection, the medium was replaced, and the supernatant was collected at 48 h and 72 h, filtered through 0.45 μm, concentrated with 8% PEG8000 at 4 °C for 12 h, and centrifuged at 4000 × g for 30 min; S44. Adjust the concentrated virus to an MOI of 10-20, add 8 μg / mL polybrene to enhance infection, and replace the culture medium containing a virus inactivator after incubation for 12 hours.

5. The method for constructing a sika deer immortalized renal epithelial cell line according to claim 1, characterized in that: The lentiviral vector of the SV40 large T antigen gene used for transfection was constructed with EF1α-SV40-IRES-puromycin and carried a resistance gene. Approximately 1×10 5 After 24 hours of adherence, 1 mL of complete culture medium and 20 μL of viral supernatant were added to the cells for infection at an MOI of about 10 to 20 for 12 hours. Fresh culture medium was then replaced and the cells were subcultured into T25 flasks for expansion.

6. The method for constructing an immortalized sika deer renal epithelial cell line according to claim 1, characterized in that: 0.05 million untransfected primary cells were plated in a 24-well plate and treated with 1-7 μg / mL puromycin. The culture medium was changed every 2 days. The lowest concentration that could kill all control cells within 4 days was 3 μg / mL. This concentration was used for continuous screening for 4 days to obtain drug-resistant cells.

7. The method for constructing an immortalized sika deer renal epithelial cell line according to claim 1, characterized in that: The drug-resistant cells were passaged for 30 to 60 generations, and the following characterizations were performed at the 10th, 35th, and 60th generations, respectively: Morphology: Under phase contrast microscopy, cells are arranged in a typical cobblestone-like pattern; Karyotype: Giemsa staining detected 2n = 66 normal chromosomes; SV40 LT gene expression: RT-qPCR Ct value <25, Western blot relative expression level was significantly higher than that of primary cells; Growth curve: measured by CCK-8 method, the cells entered the logarithmic growth phase on the 3rd to 4th day, reached the peak on the 5th day, and cell death was observed on the 6th day; Apoptosis rate: <5% detected by Annexin V / PI double staining flow cytometry.

8. The method for constructing an immortalized sika deer renal epithelial cell line according to claim 7, characterized in that: Detection of SV40 LT gene expression by RT-PCR: Total RNA was extracted from cells and analyzed by PrimeScript TM Reverse transcription and PCR amplification were performed using the FAST RT kit. The SV40 LT primer sequences used were F: ttc aga gca gaa ttg tgg agt g, and R: cct ggc gtgctt cat cat cat c. The amplified product was only detected in immortalized cells of passage 30 and above but not in primary cells.

9. Application of a method for constructing an immortalized sika deer renal epithelial cell line in establishing an in vitro model of sika deer kidney disease.

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