Human immortalized simple renal cyst cell line SRC-X1 and its application

Abstract

The application belongs to the field of microbial animal cell lines, and relates to a human immortalized simple renal cyst cell line SRC-X1 and an application thereof. The human immortalized simple renal cyst cell line is named SRC-X1, the cell line has been preserved in the China Center for Type Culture Collection on September 19, 2025, and the preservation number is CCTCC NO: C2025303. The application of the human immortalized simple renal cyst cell line SRC-X1 in preparation, screening or evaluation of renal cyst treatment drugs. The application of the human immortalized simple renal cyst cell line SRC-X1 as a renal cyst cell model. The application of the human immortalized simple renal cyst cell line SRC-X1 as experimental material in renal cyst basic or clinical research.

Description

Technical Field

[0001] This invention belongs to the field of microbial and animal cell line technology, specifically relating to a human immortalized simple renal cyst cell line SRC-X1 and its applications. Background Technology

[0002] Simple renal cysts are an acquired cystic kidney disease. These cysts typically present as thin-walled cystic structures within the renal cortex and renal pelvis. The frequency of bilateral renal cysts increases with age. While rare in childhood, the incidence rises significantly after age 30, with at least half of people over 50 having renal cysts. Prevalence varies by age, sex, and race. Approximately 8% of patients with simple renal cysts develop symptoms requiring intervention. Currently, symptomatic simple renal cysts are primarily treated with surgical removal of the cyst wall or interventional drainage of the fluid. For early-stage, small cysts that have not yet shown symptoms, a passive approach of dynamic observation is necessary, and there are currently no effective drugs for treating renal cysts. The lack of research models has slowed progress in understanding the mechanisms of renal cysts and in developing new drugs.

[0003] Human immortalized simple renal cyst cell lines serve as a bridge between basic research and clinical applications, and are irreplaceable, especially in drug screening and mechanism research. Their application value will be further expanded in the future with the development of organoid and 3D culture technologies.

[0004] However, no human immortalized simple renal cyst cell line has been reported in the literature to date. Therefore, there is an urgent need to establish a human immortalized simple renal cyst cell line. Summary of the Invention

[0005] The purpose of this invention is to address the current situation of human immortalized simple renal cyst cell lines by providing the human immortalized simple renal cyst cell line SRC-X1 and its applications, thus providing a research model for medical research on renal cysts.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a human immortalized simple renal cyst cell line named human immortalized simple renal cyst cell line (homosapiens) SRC-X1. This cell line was deposited on September 19, 2025 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: C2025303.

[0008] Establishment of the cell line: This invention uses a surgically removed renal cyst specimen from a 67-year-old woman. After digestion with a mixture of type II dispersase and type IV collagenase, the specimen is cultured in primary culture. Using standard cell culture techniques and SV40 lentivirus infection technology, an immortalized simple renal cyst cell line was successfully cultured and named SRC-X1.

[0009] The SRC-X1 cell line has the following biological characteristics:

[0010] 1. Cell growth characteristics: The cells adhere to the wall and are mainly short spindle-shaped.

[0011] 2. Cell doubling time: 29h.

[0012] 3. The cells were confirmed to be a novel human cell line by detection of short tandem repeat (STR) sequences.

[0013] 4. Immunohistochemical results showed that SRC-X1 is a kidney-derived cell line.

[0014] 5. Cells infected with SV40 lentivirus acquire immortalization capabilities, enabling them to transcribe SV40 mRNA and translate SV40 protein.

[0015] The SRC-X1 human immortalized simple renal cyst cell line can be used as a cell model for drug screening and mechanism research.

[0016] The human immortalized simple renal cyst cell line SRC-X1 can be used in the preparation, screening or evaluation of drugs for the treatment of renal cysts;

[0017] The human immortalized simple renal cyst cell line SRC-X1 can be used as a renal cyst cell model.

[0018] The human immortalized simple renal cyst cell line SRC-X1 can be used as an experimental material in basic or clinical research on renal cysts.

[0019] Application of the human immortalized simple renal cyst cell line SRC-X1 in the study of the physiological and pathological mechanisms of renal cysts.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention constructs a novel human immortalized simple renal cyst cell line, SRC-X1, providing a new tool for the study of renal cyst diseases and helping to advance the research progress of renal cyst diseases. Attached Figure Description

[0022] Figure 1 Morphological observation of SRC-X1 cells under a 40x microscope.

[0023] Figure 2 Morphological observation of SRC-X1 cells under a 200x microscope.

[0024] Figure 3 HE staining results for SRC-X1 cells.

[0025] Figure 4 The results are for SRC-X1 cell immunohistochemistry. In the diagram, A indicates positive expression of CK7 in cells; B indicates negative expression of E-cadherin in cells; C indicates negative expression of Vimentin in cells; and D indicates positive expression of Pax-2 in cells.

[0026] Figure 5 The level of SV40 mRNA in SRC-X1 cells was measured. Compared with primary cells, a significant increase in SV40 mRNA expression was observed in SRC-X1 cells. P <0.0001), indicating that the SV40 gene is integrated into SRC-X1 cells and can be transcribed into mRNA, which proves the success of cell immortalization at the gene level.

[0027] Figure 6 Images show indirect immunofluorescence of SRC-X1 cells and primary cells. In the images, A represents the detection of SRC-X1 cell nuclei (blue) using 4',6-diamidinyl-2-phenylindole (DAPI); B represents the detection of SV40 protein expression in SRC-X1 cells (green) using mouse anti-SV40 mAb; C represents the detection of primary cells with simple renal cysts (blue) using DAPI; and D represents the detection of SV40 protein expression in primary cells with simple renal cysts (green) using mouse anti-SV40 mAb.

[0028] Figure 7 The graph shows the growth curve of SRC-X1 cells. The x-axis represents cell culture time, and the y-axis represents cell number. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments will be used in conjunction with the accompanying drawings to further illustrate the invention. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Rather, this invention covers any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of the invention as defined in the claims.

[0030] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0031] Establishment and identification of SRC-X1, a human immortalized simple renal cyst cell line.

[0032] I. Establishment of the human immortalized simple renal cyst cell line SRC-X1

[0033] Clinically collected surgically removed renal cystic tissue was digested with mixed enzymes and then cultured in primary culture. This successfully established an immortalized renal cyst cell line capable of continuous passage, which has now reached the 30th generation and maintains stable cell characteristics.

[0034] This invention utilizes a renal cyst skin specimen surgically removed from a 67-year-old female patient. After digestion with a mixture of type II dispersant enzyme and type IV collagenase, primary culture was performed. Using cell culture techniques and viral infection, an immortalized renal cyst cell line was established and named the human immortalized simple renal cyst cell line (homo sapiens) SRC-X1. It was deposited on September 19, 2025, at the China Center for Type Culture Collection (CCTCC, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China), with accession number CCTCC NO: C2025303.

[0035] II. Detection of biological characteristics of the human immortalized simple renal cyst cell line SRC-X1

[0036] 1. Cell morphology observation

[0037] After the cells stabilized and were passaged, live cell observation was performed. The morphological observation of SRC-X1 cells under a microscope is shown in the image below. Figure 1 and 2 As shown. Among them, Figure 1 Cell morphology under low magnification. Figure 2 The results show that the cells are arranged in an epithelial-like pattern under a microscope, adherent to the cell wall, and are predominantly short spindle-shaped.

[0038] 2. HE and immunohistochemical staining

[0039] SRC-X1 cells in logarithmic growth phase were trypsinized and seeded onto sterile glass slides for growth. After 48 hours, the culture medium was removed, the slides were washed twice with PBS, fixed with 4% paraformaldehyde solution for 15 minutes, and then air-dried in a ventilated area. After air-drying, the slides were permeated with 0.5% Triton X-100 solution for 20 minutes and then stained with hematoxylin and eosin (HE). The results are as follows: Figure 3 As shown. Figure 3 The SRC-X1 cells were predominantly short spindle-shaped, relatively uniform in size, with large, deeply stained nuclei.

[0040] Immunohistochemical staining: After dewaxing and rehydration, slides were immersed in 10 mmol / L sodium citrate solution (pH 6.0), boiled for 90 seconds, and incubated in 3% hydrogen peroxide solution at 37°C for 15 minutes. Then, 100 μL of normal goat serum was added, and the slides were blocked at 37°C for 15 minutes to reduce non-specific antibody binding and lower background staining. The slides were then incubated with anti-CK7, anti-Pax-2, anti-E-cadherin, and anti-Vimentin at 37°C for 12 hours, respectively, followed by incubation with secondary antibody at room temperature for 50 minutes. DAB staining kit was used for color development. After rinsing the slides with running water for 5 minutes, hematoxylin was used for counterstaining, followed by graded ethanol dehydration, xylene clearing, and mounting with neutral resin. The slides were then observed under an inverted microscope (Olympus, IX73+DP74). Results are as follows: Figure 4 As shown. Figure 4 In this context, A indicates that CK7 is positively expressed in cells, and the staining site is located in the cytoplasm. Figure 4 In this context, B indicates that E-cadherin is negatively expressed in cells; Figure 4 The C in the figure indicates that Vimentin is negatively expressed in cells; Figure 4 The "D" indicates that Pax-2 is positively expressed in the cell, and the staining site is located in the cell nucleus.

[0041] 3. Identification of SV40 gene expression in cells

[0042] Total RNA was extracted from newly isolated primary cells (expanded for 5 days) and SRC-X1 cells passaged to the 15th generation, and the RNA was reverse transcribed to synthesize cDNA. The mRNA expression level of the SV40 target gene in primary and SRC-X1 cells was detected using real-time quantitative polymerase chain reaction (RT-qPCR), with GAPDH as an internal control. Primer sequences are shown in Table 1. Real-time quantitative qPCR was performed using a Mastercycler Nexus GSX1 gradient PCR instrument (Eppendorf, Germany) with TBGreen dye. The qPCR reaction system was: 100 ng DNA template, 2×TBGreen... Premix Ex Taq II. Fast qPCR: 12.5 μL of 10 μmol / L upstream primer, 1 μL of 10 μmol / L downstream primer, and sterile water to a final volume of 25 μL per tube. Reaction conditions were as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 10 s, annealing at 60℃ for 30 s, for a total of 40 cycles. Follow the 2- △△Ct The method was used for relative quantitative analysis, and the results are as follows: Figure 5As shown, compared with primary cells, a significant increase in the mRNA expression level of SRC-X1SV40 can be observed ( P <0.0001), indicating that the SV40 gene is integrated into SRC-X1 cells and can be transcribed into mRNA, which proves the success of cell immortalization at the gene level.

[0043] Table 1 Primer sequences for the target gene

[0044]

[0045] Note: SV40: simian vacuolating virus 40; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

[0046] 4. Indirect immunofluorescence identification

[0047] Primary cells in logarithmic growth phase and SRC-X1 cells passaged to the 25th generation were seeded in 12-well plates. Once the cells reached a monolayer, the culture medium was discarded, and the cells were washed three times with PBS. They were then fixed with 4% paraformaldehyde at 4°C for 30 min, followed by the addition of 0.5 mL of 0.25% Triton X-100 permeabilizer and incubation at room temperature for 10 min. The cells were then washed three times with PBS containing 1% BSA. After incubation with mouse anti-SV40 mAb at room temperature for 2 h, the cells were washed three times with PBS containing 1% BSA. Each well was then incubated with 1:50 diluted Alexa Fluor® 488 fluorescein-labeled goat anti-mouse IgG antibody and a final concentration of 5 μg / mL DAPI solution, and incubated at room temperature in the dark for 2 h. The cells were washed three times with PBS containing 1% BSA, and 0.5 mL of PBS was added to each well. The cells were observed and photographed under a fluorescence microscope. The results are shown below. Figure 6 As shown. From Figure 6 It can be seen that the nuclei of SRC-X1 cells were stained with DAPI ( Figure 6 A in the sample is blue and has been stained with SV40 antibody. Figure 6 B) showed green fluorescence (positive expression of SV40 protein); while primary cells showed only blue fluorescence in the nucleus after DAPI staining. Figure 6 In the C), primary cells showed no green fluorescence after SV40 staining ( Figure 6 The presence of negative expression of SV40 protein in D cells demonstrated that SRC-X1 cells stably express SV40 protein, while the primary cell control group did not express SV40 protein. As a result, SRC-X1 cells were identified at the protein level.

[0048] 5. STR testing

[0049] SRC-X1 cells in the logarithmic growth phase were harvested, digested with trypsin, and sent to Suzhou Jianda Biotechnology Co., Ltd. for STR analysis, confirming that SRC-X1 cells were a novel human cell line. The results are shown in Table 2.

[0050] Table 2. Overview of SRC-X1 Cell STRs

[0051]

[0052] The STR data in Table 2 show that the SRC-X1 cell line is a novel human cell line with no cross-contamination with cells in existing cell line databases.

[0053] 6. Determination of cell doubling time

[0054] SRC-X1 cells in the logarithmic growth phase were selected and digested with 0.25% trypsin at 37°C for 5 min. Three volumes of complete culture medium (composed of RPMI-1640 medium and 10% fetal bovine serum) were added, and the cells were collected by centrifugation. The cells were then resuspended in an appropriate amount of complete culture medium to prepare a single-cell suspension. The prepared single-cell suspensions were counted, and cells of 0 and 2.5 × 10⁻⁶ cells were counted. 3 4×10 3 7.5×10 3 1×10 4 1.25×10 4 1.5×10 4 1.75×10 4 Cells were seeded in 96-well plates, with 6 replicates per group, and incubated at 37°C in a 5% CO2 incubator. At 0, 24, 48, 72, and 96 h, 110 μL / well of CCK8 working solution (RPMI-1640:CCK8 = 10:1) was added. After 2 h of incubation, the absorbance of each well was measured at 450 nm. Growth curves of SRC-X1 cells were plotted using GraphPad Prism 8.0.2, with cell number on the x-axis and OD value on the y-axis, and the cell doubling time was calculated. The cell doubling time was approximately 29 h. The growth curves of SRC-X1 cells are shown below. Figure 7 As shown. Figure 7 It clearly shows the growth trend of cells at different time points, providing intuitive data support for a deeper understanding of cell proliferation characteristics.

[0055] The above embodiments are merely preferred embodiments of the present invention and should not be considered as limiting the scope of the present invention. All equivalent variations and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A human immortalized simple renal cyst cell line, characterized in that... It is named the human immortalized simple renal cyst cell line (homo sapiens) SRC-X1. This cell line was deposited at the China Center for Type Culture Collection on September 19, 2025, with accession number CCTCC NO: C2025303.

2. The use of the human immortalized simple renal cyst cell line as described in claim 1 in screening or evaluating drugs for the treatment of renal cysts.

3. The application of the human immortalized simple renal cyst cell line as described in claim 1 as a renal cyst cell model.

4. The application of the human immortalized simple renal cyst cell line as described in claim 1 as experimental material in basic or preclinical research on renal cysts.

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