Col4a5 + / -mouse immortalized podocyte line and application thereof
By establishing an immortalized mouse podocyte cell line through lentiviral infection, the problem of difficult cell line construction in existing technologies has been solved, and stable passage and efficient culture have been achieved, supporting accurate pathological modeling and drug development for Alport syndrome.
Patent Information
- Application Number
- CN202511271735.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-08
- Publication Date
- 2026-01-16
AI Technical Summary
Constructing Col4a5+/- cell lines using existing technologies is difficult, plasmid transfection rates are low, and primary podocyte culture cycles are long and have high failure rates, making it difficult to achieve effective in vitro AS studies.
We provide an immortalized mouse podocyte cell line of Col4a5+/-, which achieves cell immortalization through lentiviral infection, maintains the expression of Nephrin and WT-1 proteins, and is stably passaged for more than 30 generations, making it suitable for cell models and drug development for Alport syndrome.
It enables stable passage and efficient culture of Col4a5+/- cell lines, supports precise pathological modeling, provides a CRISPR-Cas9 gene correction platform, discovers new molecular targets, tests drug efficacy, and is suitable for the treatment and research of Alport syndrome.
Smart Images
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biology and medical technology, in particular to a Col4a5+ / - mouse immortalized podocyte cell line and its use. BACKGROUND
[0002] Alport syndrome (AS) is the second most common type of genetic kidney disease, with an incidence of about 1 / 5000-1 / 53000, and is the second leading cause of genetic renal failure. Its pathogenesis is the basement membrane structural defect caused by mutations in genes encoding collagen type IV alpha 3-alpha 5 chains.
[0003] The genetic patterns of AS include three types: X-linked dominant inheritance (XLAS), autosomal recessive inheritance (ARAS), and autosomal dominant inheritance (ADAS). X-linked dominant inheritance accounts for 80-85%, caused by mutations in the COL4A5 gene, and the proportion of male patients progressing to end-stage renal disease before the age of 30 is 100%; autosomal recessive inheritance (ARAS) accounts for about 15%, requires double allelic mutations in COL4A3 / COL4A4, and is characterized by early and severe kidney disease; autosomal dominant inheritance (ADAS) accounts for <5%, and single allelic mutation can cause disease, with a milder phenotype.
[0004] Renin-angiotensin-aldosterone system blockers (RAASi) are currently the first recommended drug to delay the progression of Alport syndrome kidney disease, but they can only delay the occurrence of kidney disease to a certain extent, and most of them progress to end-stage kidney disease at a later stage. There is no radical or etiological treatment, so better methods for treating Alport syndrome are urgently needed.
[0005] Previous in vitro studies of AS required transfection of plasmids into podocytes to reduce their expression, or direct extraction of primary podocytes from AS mice for culture. However, both of the above methods have drawbacks, including low transfection efficiency, limited reduction of Col4a5 expression, and inability to obtain a Col4a5+ / - cell line. Direct extraction of primary podocytes from AS mice for culture has a long culture period, high failure rate, and limited growth period of primary podocytes, requiring repeated extraction.
[0006] The above shortcomings have seriously affected in vitro studies of AS, and a new cell for in vitro studies of AS is needed. SUMMARY
[0007] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a Col4a5+ / - mouse immortalized podocyte cell line and its use, to solve the problems in the prior art.
[0008] To achieve the above object and other related objects, the present application provides a Col4a5+ / - mouse immortalized podocyte cell line, which has a preservation number of CGMCC NO. 46563.
[0009] The Col4a5+ / - mouse immortalized podocyte cell line is a Col4a5+ / - gene-deficient mouse immortalized kidney podocyte cell line.
[0010] The Col4a5+ / - mouse immortalized podocyte cell line expresses Nephrin protein and WT-1 protein.
[0011] The present application also provides use of the Col4a5+ / - mouse immortalized podocyte cell line in preparation of a genetic kidney disease cell model.
[0012] Preferably, the genetic kidney disease is Alport syndrome.
[0013] Preferably, the genetic kidney disease cell model prepared by using the Col4a5+ / - mouse immortalized podocyte cell line can be used for development or screening of a therapeutic drug or a diagnostic drug for genetic kidney disease.
[0014] Preferably, the genetic kidney disease cell model is a cell model simulating a female suffering from genetic kidney disease.
[0015] As described above, the Col4a5+ / - mouse immortalized podocyte cell line and use thereof of the present application have the following beneficial effects: strong stability guarantee, expression of podocyte markers (Nephrin / WT-1) > 90% after 30 generations of continuous passage, typical podocyte structure integrity can still be observed under an electron microscope, and the cell line can be cultured and passed in large quantities, the culture requirements are relatively simple, a large number of experimental cells can be obtained in a short time, the overall experimental period is short, and the cell line is suitable for promotion. The cell line has the following applications:
[0016] 1) Overcome the technical difficulties of single allele mutation (Col4a5+ / -) cell line construction in the prior art, such as low transfection efficiency and allele loss, which can be used for precise pathological modeling, and for the first time to simulate the COL4A5 gene dose effect, Col4a5+ / - cell line corresponds to the phenotype of female XLAS carrier with mild disease; support the research of type IV collagen triple helix assembly: through the expression of α3-α4-α5 chain of cell line, reveal the key node of basement membrane structure defect; 2) Can be used as a molecular mechanism analysis platform for CRISPR-Cas9 gene correction experiment, can quickly construct isogenic control line (such as repair COL4A5 mutation); can find new target points of podocyte-basement membrane bidirectional signal transduction through transcriptome and proteome analysis. 3) Can be used for drug testing: as a cell model, test whether traditional Chinese medicine such as triptolide can increase the expression of Col4a5 protein in immortalized Col4a5+ / - podocytes, and then possibly be used for the treatment or research of Alport syndrome to alleviate the endoplasmic reticulum stress mechanism of immortalized Col4a5+ / - podocyte line; used to evaluate the effect of gene therapy, for example, as a benchmark for Col4a5 protein expression level, quantitatively detect the protein expression recovery level of Col4a5 after Col4a5 gene therapy such as AAV treatment. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 The micrograph shows Col4a5+ / - mouse primary kidney podocytes, the left panel is magnified 100 times, and the right panel is magnified 200 times.
[0018] Figure 2 The micrograph shows Col4a5+ / - mouse immortalized kidney podocytes, the left panel is magnified 100 times, and the right panel is magnified 200 times.
[0019] Figure 3 The micrograph shows Col4a5+ / - mouse immortalized kidney podocytes, the left panel is magnified 100 times, and the right panel is magnified 200 times.
[0020] Figure 4 The micrograph shows Col4a5+ / - mouse immortalized kidney podocytes, the left panel is magnified 100 times, and the right panel is magnified 200 times.
[0021] Figure 5 The micrograph shows the protein expression of Col4a5+ / - mouse immortalized podocyte line after treatment with triptolide. DETAILED DESCRIPTION
[0022] The application provides a Col4a5+ / - mouse immortalized podocyte cell line, which has a preservation number of CGMCC NO. 46563.
[0023] The Col4a5+ / - mouse immortalized podocyte cell line has been preserved, and is classified and named as a mouse podocyte cell line, has a preservation number of CGMCC NO. 46563, a preservation date of July 24, 2025, and a preservation unit name of China General Microbiological Culture Collection Center (CGMCC), a preservation unit abbreviation of CGMCC, and a preservation unit address of No. 1 Yard 3, North Chenxi Road, Chaoyang District, Beijing, China Institute of Microbiology.
[0024] The Col4a5+ / - mouse immortalized podocyte cell line is a Col4a5+ / - gene-deficient mouse immortalized kidney podocyte cell line.
[0025] Immortalization refers to a state in which a cell cultured in vitro has the ability of unlimited proliferation under the influence of the outside world.
[0026] The mouse immortalized podocyte cell line can be subcultured for more than 30 generations.
[0027] In some embodiments of the application, the Col4a5+ / - mouse immortalized podocyte cell line comprises primary cells or daughter cells thereof.
[0028] The Col4a5+ / - mouse immortalized podocyte cell line can express Nephrin protein and WT-1 protein, and can stably proliferate and be subcultured. After being subcultured for 30 generations, the expression rate of podocyte markers (Nephrin / WT-1) of the Col4a5+ / - mouse immortalized podocyte cell line is still higher than 90%, and the typical podocyte structure integrity can still be observed under an electron microscope.
[0029] The Genbank number of the mouse Nephrin protein is NC_000073. The Genbank number of the mouse WT-1 protein is NC_000068.
[0030] The Col4a5+ / - mouse immortalized podocyte cell line corresponds to the phenotype of a female X-linked dominant (XLAS) carrier, and is X + X - type, and thus can realize COL4A5 gene dose effect simulation.
[0031] The application also provides use of the Col4a5+ / - mouse immortalized podocyte cell line in preparation of a genetic kidney disease cell model.
[0032] In some embodiments of the present application, the genetic kidney disease is Alport Syndrome (AS).
[0033] Alport Syndrome is a syndrome characterized by hematuria, proteinuria, and progressive reduction in renal function, and some patients have extrarenal manifestations such as sensorineural hearing loss (SNHL) and ocular lesions. This disease is caused by mutations in COL4A3, COL4A4 and COL4A5 genes that encode the α3α4α5 chain of type IV collagen. According to different genetic patterns, it is divided into X-linked Alport syndrome (XLAS), autosomal recessive Alport syndrome (ARAS), autosomal dominant Alport syndrome (ADAS) and digenic Alport syndrome proposed in recent years. Different genetic patterns of Alport syndrome have genotypic and phenotypic heterogeneity, but most Alport syndrome patients will progress to end-stage kidney disease (ESKD), which is one of the main genetic kidney diseases leading to renal failure.
[0034] XLAS is caused by pathogenic variants in COL4A5 gene and is the most common genetic pattern. The risk of male patients progressing to ESKD can be as high as 100%, and the age of progression to ESKD and extrarenal manifestations is related to the COL4A5 genotype. The risk of female patients progressing to ESKD is about 25%, and the risk of progression to ESKD increases with age. Risk factors for disease progression include a history of gross hematuria in childhood, progressive worsening of proteinuria, and SNHL. A small number of XLAS patients have large deletions involving the 5' end of the COL4A5 gene to the 2nd intron of the adjacent COL4A6 gene, and the clinical manifestations of patients can be combined with leiomyoma.
[0035] In some embodiments of the present application, the genetic kidney disease cell model prepared by using the Col4a5+ / - mouse immortalized podocyte cell line can be used for the development or screening of therapeutic drugs or diagnostic drugs for genetic kidney diseases.
[0036] In some embodiments of the present application, the genetic kidney disease cell model is a female genetic kidney disease cell model.
[0037] The cell line of the present application is used for non-diagnostic or therapeutic purposes. It can be used for basic research.
[0038] The advantages and effects of the present application can be easily understood by those skilled in the art from the above description. The present application can also be implemented or applied by different specific embodiments, and the details in the description can be modified or changed based on different views and applications without departing from the spirit of the present application.
[0039] Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present application are used to describe the specific embodiments, rather than to limit the scope of protection of the present application; in the specification and claims of the present application, the singular forms "a", "an" and "the" include the plural forms unless otherwise explicitly stated in the text.
[0040] When the embodiments give numerical ranges, it should be understood that, unless otherwise stated by the present application, both endpoints of each numerical range and any number between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art. In addition to the specific methods, devices, materials used in the embodiments, any method, device and material of the prior art similar or equivalent to the methods, devices and materials described in the embodiments of the present application can also be used to implement the present application according to the mastery of the prior art by those skilled in the art and the description of the present application.
[0041] Example 1, Isolation and culture of mouse kidney podocytes
[0042] 1.1 Experimental materials
[0043] DMEM / F12 medium, FBS (Gibco); rat tail collagen (Sigma); penicillin-streptomycin (PS, Shanghai Biyun Tian); CO2 incubator (Thermo).
[0044] C57BL / 6Smoc-Col4a5em1(R471X)Smoc mice were purchased from Shanghai South Model Organism Technology Co., Ltd., and the offspring mice were obtained after mating with WT mice.
[0045] 1.2 Experimental steps
[0046] 1) Differential filtration method to isolate glomerulus: female offspring mice were sacrificed under anesthesia, both kidneys were removed under sterile conditions and placed in pre-cooled DMEM / F12 medium. The kidney capsule was removed and the renal cortex was cut. The renal cortex was gently crushed on a 200\150\80um sieve with a 10ml syringe needle, and pre-cooled DMEM / F12 medium was used to rinse from time to time. The cell suspension was collected and filtered through a 300-mesh sieve. The glomeruli on the 300-mesh sieve were collected and observed under an inverted microscope.
[0047] 2) Glomerular implantation: DMEM / F12 + 10% FBS + 1% PS complete medium was used to resuspend the glomeruli, which were then implanted in a 10cm 2 plastic culture dish coated with rat tail collagen at 37°C in a CO2 incubator.
[0048] As Figure 1 shown, Col4a5+ / - mouse primary podocytes were obtained by crawling out of the glomerulus. The results showed that the cell morphology was clear and complete, and the typical foot process structure integrity was observed under electron microscope.
[0049] Example 2: Immortalization of mouse kidney podocytes
[0050] 2.1 Experimental materials
[0051] Puromycin, Polybrene (Sigma); 0.25% trypsin (Shanghai Biyun Tian); CO2 incubator (Thermo).
[0052] 2.2 Experimental steps
[0053] 1. After 10 days of in vitro culture of primary mouse kidney podocytes, the cells were washed with PBS 3 times, digested with 0.25% trypsin in the incubator for 3 minutes, and then the cell suspension was filtered through a 40uM cell sieve. The cell suspension was centrifuged at 1200rpm for 5min, and finally the cell pellet was resuspended with complete medium and plated in a 6cm culture dish coated with rat tail collagen;
[0054] 2. When the above cells grew to 50% confluence, they were infected with slow virus HBLV-SV40T-PURO (from Hanheng Biotechnology (Shanghai) Co., Ltd.) MOI = 80 (containing 5ug / mL Polybrene to enhance the efficiency of slow virus infection) in a 37°C, 5% CO2 incubator overnight;
[0055] 3. After 2 days of slow virus infection, the medium was changed, and then the culture was continued for another 4 days, with medium change every 2 days;
[0056] 4. Add 2 ug / mL Puromycin in the complete medium, and continue to culture for 96h;
[0057] 5. When the cells grow to confluence, continue to culture the cells by routine subculture.
[0058] The first generation of immortalized mouse kidney podocytes were subcultured for several times, and the cells were in good condition. As shown in Figure 2 , the immortalized kidney podocytes of the 3rd generation of Col4a5+ / -mice.
[0059] Example 3 Immunofluorescence staining identification
[0060] Nephrin and WT-1 are both marker proteins of kidney podocytes, so these two antibodies were used to identify whether the obtained immortalized cells were kidney podocytes. The steps are as follows:
[0061] (1) When the immortalized mouse kidney podocytes grow to 80% confluence, discard the culture medium, rinse the cells with warm PBS for 2 times, 10 min each time, and then fix the cells with 4% paraformaldehyde at room temperature for 15 min;
[0062] (2) Rinse the cells with PBS for 2 times, 10 min each time, and then permeabilize the cells with 0.1% Triton X-100 at 4°C for 15 min;
[0063] (3) Rinse the cells with PBS for 2 times, 10 min each time, and then block the cells with 4% BSA at room temperature for 30 min;
[0064] (4) Dilute the Nephrin antibody (Beijing Boao Nephrin bs-4866R) and the WT-1 antibody (Beijing Boao bs-6983R) at a ratio of 1:100, and then incubate them in a 4°C refrigerator overnight;
[0065] (5) Rinse the cells with PBS for 3 times, 10 min each time, dilute the anti-CY3 labeled secondary antibody at a ratio of 1:100, and place it at 37°C for 1 h;
[0066] (6) Rinse with PBS for 3 times, 10 min each time, and observe the images under an inverted fluorescence microscope and take pictures.
[0067] The immunofluorescence staining of Col4a5+ / -cells is shown in Figure 3 and Figure 4 The results of immunofluorescence staining suggest that Col4a5+ / -cells express Nephrin and WT-1, which are kidney podocytes.
[0068] Example 4
[0069] Col4a5+ / - mouse immortalized podocyte cell line and Col4a5+ / + cells were cultured in DMEM / F12 + 10% FBS + 1% PS medium at 37°C. The treatment group was treated with 10 ng / ml of Triptolide (TCI, T2899) for 30 minutes to treat podocyte injury, and the control group was treated with the same dose of PBS. After treatment, the cells were lysed for Western Blot to detect the expression of Col4a5 protein and endoplasmic reticulum stress indicator protein (p-eIF2a / eIF2a).
[0070] The results are shown in Figure 5 After treatment with Triptolide, the expression of Col4a5 protein was increased, and the endoplasmic reticulum stress indicator (p-eIF2a / eIF2a) in the Col4a5+ / - mouse immortalized podocyte cell line was increased, and the protein expression was down-regulated after Triptolide intervention. Based on this, it can be explained that in the Col4a5+ / - podocytes, Triptolide can increase the expression of Col4a5 protein and relieve endoplasmic reticulum stress, thereby providing experimental evidence for the in vitro treatment of Alport syndrome. The experiment was carried out on the Col4a5+ / - mouse immortalized podocyte cell line, which also showed the stability of the cell line, which can be repeatedly subcultured and is suitable for promotion.
[0071] The above examples are intended to illustrate the disclosed embodiments of the present application and should not be construed as limiting the present application. In addition, various modifications and changes in the method of the application listed herein are obvious to those skilled in the art without departing from the scope and spirit of the present application. Although the present application has been specifically described in conjunction with various preferred embodiments thereof, it should be understood that the present application should not be limited to these specific embodiments. In fact, various modifications such as those described above to those skilled in the art to obtain the application should be included within the scope of the present application.
Claims
1. A Col4a5+ / - mouse immortalized podocyte cell line, with a preservation number of CGMCC NO. 46563.
2. The Col4a5+ / - murine immortalized podocyte cell line of claim 1, wherein, The Col4a5+ / - mouse immortalized podocyte cell line is a Col4a5+ / - gene-deficient mouse immortalized kidney podocyte cell line.
3. The Col4a5+ / - murine immortalized podocyte cell line of claim 1, wherein, The Col4a5+ / - mouse immortalized podocyte cell line expresses Nephrin protein and WT-1 protein.
4. The Col4a5+ / - murine immortalized podocyte cell line of claim 1, wherein, The Col4a5+ / - mouse immortalized podocyte cell line comprises primary cells or progeny cells thereof. 5.Use of the Col4a5+ / - mouse immortalized podocyte cell line according to any one of claims 1 to 4 in the preparation of a genetic kidney disease cell model.
6. Use according to claim 5, characterized in that, The genetic kidney disease is Alport syndrome.
7. Use according to claim 5, characterized in that, The genetic kidney disease cell model is used for screening therapeutic drugs or diagnostic drugs for genetic kidney disease.
8. Use according to claim 5, characterized in that, The genetic kidney disease cell model is a cell model simulating a female suffering from genetic kidney disease. 9.Use of triptolide in the preparation of a therapeutic drug for genetic kidney disease.
10. Use according to claim 9, characterized in that, The genetic kidney disease is Alport syndrome; preferably, it is a COL4A5+ / - gene-deficient Alport syndrome.