Human normal kidney immortalized cell line NRC-X1 and application thereof

Abstract

The invention discloses a human normal kidney immortalized cell line NRC-X1 and application thereof, and belongs to the field of microbial animal cell lines. The human normal kidney immortalized cell line is named as NRC-X1, and the preservation number is CCTCC (China Center For Type Culture Collection) NO: C2025168. The invention also discloses the application of the human normal kidney immortalized cell line NRC-X1 in a virus infection mechanism. The invention also discloses application of the human normal kidney immortalized cell line NRC-X1 in research of drug renal toxicity evaluation. The invention also discloses the application of the human normal kidney immortalized cell line NRC-X1 in bioartificial kidney and cell therapy. The invention also discloses application of the human normal kidney immortalized cell line NRC-X1 in research of kidney physiological and pathological mechanisms.

Description

Technical Field

[0001] This invention belongs to the field of microbial animal cell lines, specifically relating to a human normal kidney immortalization cell line NRC-X1 and its applications. Background Technology

[0002] The kidneys are vital organs in the human body. Their primary function is to produce urine, thereby removing metabolic waste products and certain toxins from the body. Simultaneously, through reabsorption, they retain water and other useful substances such as glucose, protein, amino acids, sodium ions, potassium ions, and bicarbonate, regulating water and electrolyte balance and maintaining acid-base balance. The kidneys also have endocrine functions, producing renin, erythropoietin, active vitamin D3, prostaglandins, and kinins. They also serve as a site for the degradation of some endocrine hormones and a target organ for extrarenal hormones. These functions of the kidneys ensure the stability of the internal environment and allow for normal metabolism.

[0003] The kidneys have a strong compensatory ability. When more than 75% of the kidneys are damaged, the remaining kidney tissue can still maintain normal physiological functions for a period of time through compensation. When the kidneys continue to be damaged, metabolic waste products cannot be excreted from the body, directly threatening life.

[0004] Immortalized human normal kidney cells serve as a bridge between basic research and clinical applications, and are irreplaceable, especially in drug development, toxicity testing, and disease mechanism research. With the development of organoid and 3D culture technologies, their application value will be further expanded.

[0005] Currently, the immortalized human kidney cell lines used in medical research are: HK-2, human renal proximal tubular epithelial cells (SV40 immortalized), commonly used for toxicity studies; RPTEC / TERT1, telomerase-immortized human renal proximal tubular cells, retaining characteristics closer to primary cells; and HEK293, human embryonic kidney cells (adenovirus transfected), widely used in molecular biology and viral packaging. However, to date, no literature reports on immortalized normal Chinese kidney cell lines. Therefore, establishing immortalized normal Chinese kidney cell lines is essential. Summary of the Invention

[0006] The purpose of this invention is to address the current lack of Chinese-derived normal kidney immortalization cell lines by providing a Chinese-derived normal kidney immortalization cell line, NRC-X1, and its applications, thus offering a research model for Chinese kidney medicine research.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The present invention provides a human normal kidney immortalization cell line named human normal kidney immortalization cell line NRC-X1, which has been deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: C2025168.

[0009] Establishment of the cell line: This invention uses normal kidney tissue from a surgical resection specimen of renal pelvis cancer from a 70-year-old female patient. After digestion with a mixture of type II collagenase and neutral protease, it is cultured in primary culture. Using standard cell culture techniques and SV40 large T antigen gene transfection technology, a kidney-derived immortalized cell line was successfully cultured and named NRC-X1.

[0010] This cell line was deposited on May 29, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: C2025168.

[0011] This cell line has the following biological characteristics:

[0012] 1. Cell growth characteristics: Cells adhere to the wall and are mainly short spindle-shaped.

[0013] 2. Cell doubling time: 30h.

[0014] 3. STR testing confirmed that the cells were a new human cell line.

[0015] 4. Immunohistochemical results showed that NRC-X1 is a kidney-derived cell line.

[0016] 5. Cells transfected with SV40 acquire immortalization capabilities, enabling them to transcribe SV40T mRNA and translate SV40T protein.

[0017] Application of the human normal kidney immortalized cell line NRC-X1 in viral infection mechanisms.

[0018] The application of the human normal kidney immortalized cell line NRC-X1 in the evaluation of drug nephrotoxicity.

[0019] Application of the human normal kidney immortalized cell line NRC-X1 in bioartificial kidney and cell therapy.

[0020] Application of the human normal kidney immortalized cell line NRC-X1 in the study of kidney physiological and pathological mechanisms.

[0021] The application of the human normal kidney immortalized cell line NRC-X1 in the preparation of kidney disease research models utilizes the normal kidney cell biological characteristics of this cell line to simulate the physiological or pathological state of the kidney, and to study the pathogenesis, drug targets and efficacy evaluation of kidney diseases.

[0022] The application of the human normal kidney immortalized cell line NRC-X1 in drug screening and toxicology studies allows for the evaluation of drug nephrotoxicity or therapeutic efficacy by detecting the effects of drugs on cell proliferation, biomarker expression, or function, thus providing an in vitro model for the development of kidney-related drugs.

[0023] The human normal kidney immortalized cell line NRC-X1 is used in cell biology research to study the mechanisms of kidney cell proliferation, differentiation, and apoptosis, or as a control cell line to compare the molecular characteristics of abnormal kidney cells.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention constructs a novel human normal kidney immortalized cell line, NRC-X1, derived from Chinese individuals. This NRC-X1 cell line can serve as a cell model for drug development, nephrotoxicity testing, disease mechanism research, and bioartificial kidney research. It provides a novel tool for kidney disease research and contributes to advancing research progress in kidney diseases in China. Attached Figure Description

[0026] Figure 1 This is a morphological observation of NRC-X1 cells under a microscope.

[0027] Figure 2 HE staining results for NRC-X1 cells.

[0028] Figure 3 The results are for NRC-X1 cell immunohistochemistry. In the figures, A indicates positive expression of CD10 in cells; B indicates positive expression of CK7 in cells; C indicates positive expression of PAX2 in cells; D indicates positive expression of E-cadherin in cells; and E indicates positive expression of Vimentin in cells.

[0029] Figure 4 The level of SV40T mRNA in NRC-X1 cells.

[0030] Figure 5 These are indirect immunofluorescence images of NRC-X1 cells and primary cells. NRC-X1 cells expressed SV40T protein, while the primary cell control group did not express SV40T protein, thus identifying NRC-X1 cells at the protein level.

[0031] Figure 6 The graph shows the growth curve of NRC-X1 cells. The horizontal axis represents cell culture time, and the vertical axis represents cell number. Detailed Implementation

[0032] 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.

[0033] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0034] Establishment and identification of the human normal kidney immortalized cell line NRC-X1.

[0035] I. Establishment of the human normal kidney immortalized cell line NRC-X1

[0036] Normal kidney tissue was collected from surgically removed kidneys and cultured using mixed enzymes. This led to the successful establishment of a normal kidney immortalized cell line that can be continuously passaged. The line has been passaged to 40 generations and the cell characteristics remain stable.

[0037] This invention utilizes a surgical specimen obtained from a 70-year-old female patient. After digestion with a mixture of type II collagenase and neutral protease, primary culture was performed. Using cell culture techniques and viral transfection, a normal renal immortalization cell line was established and named NRC-X1. It was deposited on May 29, 2025, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), with accession number CCTCC NO: C2025168.

[0038] II. Detection of biological characteristics of the human normal kidney immortalized cell line NRC-X1

[0039] 1. Cell morphology observation

[0040] After the cells stabilized and were passaged, live-cell observation was performed. The results showed that, under a microscope, the cells exhibited an epithelial-like arrangement, adherent growth, and were predominantly short spindle-shaped. The morphological observation of NRC-X1 cells under a microscope is shown in the image below. Figure 1 As shown. Figure 1 Image A shows cell morphology under low magnification (40x). Figure 1 Image B shows the cell morphology under high magnification (200x).

[0041] 2. HE and immunohistochemical staining

[0042] NRC-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 stained with hematoxylin and eosin (HE). The results are shown below. Figure 2 As shown.

[0043] Immunohistochemical staining: After dewaxing and rehydration, the slide was immersed in 10... In a sodium citrate solution at pH 6.0, the slides were boiled for 90 seconds, incubated in 3% hydrogen peroxide solution at 37°C for 15 minutes, and then 100 μL of normal goat serum was added. The slides were then blocked at 37°C for 15 minutes to reduce non-specific antibody binding and lower background staining. Next, the slides were incubated with anti-CD10, anti-CK7, anti-PAX2, anti-E-cadherin, and anti-Vimentin at 37°C for 12 hours, respectively. Afterward, secondary antibodies were added and the slides were incubated at room temperature for 50 minutes. DAB staining was performed, and the slides were rinsed with running water for 5 minutes, counterstained with hematoxylin, dehydrated with graded ethanol, cleared with xylene, mounted with neutral resin, and observed under an inverted microscope (Olympus, IX73+DP74). Results are as follows: Figure 3 As shown.

[0044] Figure 3 In this context, A indicates that CD10 is positively expressed in cells, manifested by obvious brown staining in the cytoplasm or cell membrane; Figure 3 The B in the text indicates that CK7 is positively expressed in cells, and the staining site is located in the cytoplasm. Figure 3 The "C" indicates that PAX2 is positively expressed in cells, and the staining site is located in the cell nucleus. Figure 3 The D in the text indicates that E-cadherin is positively expressed in the cell, resulting in brown staining of the cytoplasm or cell membrane. Figure 3 The "E" indicates that Vimentin is positively expressed in the cell, and the staining site is located in the cytoplasm.

[0045] 3. Identification of SV40 gene expression in cells

[0046] Total RNA was extracted from newly isolated primary cells (expanded for 5 days) and NRC-X1 cells passaged to the 35th generation, and the RNA was reverse transcribed to synthesize cDNA. The mRNA expression level of the target gene SV40 in primary and NRC-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 the TB Green dye method. The qPCR reaction system was: 100 ng DNA template, 12.5 μL of 2×TB Green Premix Ex Taq II Fast qPCR, 10... 1 μL of upstream primer, 10 1 μL of downstream primer was added, and sterile water was added to bring the volume to 25 μL per tube. The reaction conditions were as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 10 s, and annealing at 60°C for 30 s, for a total of 40 cycles. The method was used for relative quantitative analysis, and the results are as follows: Figure 4 As shown. Figure 4 This is a bar chart showing the relative levels of SV40T mRNA in NRC-X0 (primary cells) and NRC-X1 cells. The horizontal axis represents cell type, specifically NRC-X0 and NRC-X1 (35th generation cells); the vertical axis represents the relative level of SV40T mRNA. Black bars represent NRC-X0, and gray bars represent NRC-X1. Figure 4 As can be seen, the SV40T mRNA level in NRC-X1 cells is significantly higher than that in NRC-X0 cells compared to primary cells. The error bar at the top of the column reflects the degree of data dispersion. The annotations indicate that the differences between the two groups are extremely significant (P < 0.0001). From the perspective of gene transcription, this shows that the SV40 gene was successfully integrated into NRC-X1 cells and effectively transcribed into mRNA, which proves the success of cell immortalization at the gene level.

[0047] Table 1 Primer sequences for the target gene

[0048]

[0049] Note: SV40: simian vacuolating virus 40; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

[0050] 4. Indirect immunofluorescence identification

[0051] Newly isolated primary cells (expanded for 5 days) in logarithmic growth phase and NRC-X1 cells passaged to the 35th 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. The cells 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 at room temperature for 2 h with mouse anti-SV40 monoclonal antibody, the cells were washed three times with PBS containing 1% BSA. A 1:50 dilution of [anti-SV40 antibody] was added to each well. Fluorescein-labeled goat anti-mouse IgG antibody and final concentration 5 The cells were incubated in DAPI solution at room temperature in the dark for 2 hours. 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 experiment was performed in triplicate to ensure reproducibility. Indirect immunofluorescence images of NRC-X1 cells and primary cells are shown below. Figure 5 In experiments A and C, 4',6-diamidinyl-2-phenylindole (DAPI) was used to stain the cell nucleus, producing blue fluorescence that clearly showed the nuclear structure and distribution, serving as a reference for cell localization. Experiments B and D used mouse anti-SV40T mAb to detect SV40T protein; positive expression sites showed green fluorescence. The results showed that green fluorescence was detectable in NRC-X1 cells, indicating that these cells expressed SV40T protein. In contrast, the primary cell control group showed no green fluorescence signal, indicating that the primary cells did not express SV40T protein. This allowed for the identification of the immortalization characteristics of the NRC-X1 cell line at the protein level. Furthermore, the combination of DAPI staining and SV40T protein detection results visually presented the correspondence between the expression location of the target protein within the cell and the overall cell structure.

[0052] 5. STR testing

[0053] NRC-X1 cells in logarithmic growth phase were collected after trypsin digestion and sent to Suzhou Jianda Biotechnology Co., Ltd. for STR analysis, confirming that NRC-X1 cells are a novel human cell line. The results are shown in Table 2.

[0054] Table 2. Overview of NRC-X1 Cell STRs

[0055]

[0056] STR typing results showed that the STR profile of the NRC-X1 cell line was significantly different from the typical STR profile of commonly used kidney-derived cell lines (such as HK-2, HEK293, RPTEC / TERT1), and no matching entries were found in public STR databases (such as ExPASy), confirming that the NRC-X1 cell line is a novel human-derived cell line and that there is no cross-contamination with the aforementioned common cell lines.

[0057] 6. Determination of cell doubling time

[0058] NRC-X1 cells in the logarithmic growth phase were selected and digested with 0.25% trypsin at 37°C for 5 min. Then, 3 times the volume of trypsin was added to complete culture medium (composed of RPMI-1640 medium, 10% fetal bovine serum, 1% penicillin (100 U / mL) - streptomycin (100 U / mL)). (The mixture was prepared by centrifugation, collecting the precipitate, and resuspending the cells in an appropriate amount of complete culture medium to prepare a single-cell suspension. The prepared single-cell suspension was then counted, and cells of 0 and 2.5 × 10⁻⁶ cells were counted separately.) 3 4×10 3 7.5×10 3 1×10 4 1.25×10 4 1.5×10 4 1.75×10 4 and 2×10 4 Cells were seeded in 96-well plates, with 6 replicates per group, and incubated at 37°C in a 5% CO2 incubator. After cell attachment, 110 μL of CCK8 working solution was added per well (RPMI-1640:CCK8 = 10:1), and after 2 hours of incubation, the absorbance of each well was measured at 450 nm. A standard curve was plotted with cell count on the x-axis and OD value on the y-axis. 5 × 10⁶ cells were then seeded in a 96-well plate with 6 replicates per well. 3 Cells were seeded in 96-well plates with a blank control. Absorbance at 450 nm was measured at 0, 24, 48, 72, and 96 hours. Growth curves of NRC-X1 cells were plotted using GraphPadPrism 8.0.2, with seeding time on the x-axis and cell number on the y-axis, and the cell doubling time was calculated. The cell doubling time was approximately 30 hours. The growth curves of NRC-X1 cells are shown below. Figure 6 As shown. Figure 6 It clearly shows the growth trend of cells at different time points, providing intuitive data support for a deeper understanding of cell proliferation characteristics.

[0059] 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 normal kidney immortalized cell line, characterized in that... It was named the human normal kidney immortalized cell line NRC-X1, with accession number CCTCC NO: C2025168.

2. The application of the human normal kidney immortalized cell line as described in claim 1 in the viral infection mechanism.

3. The application of the human normal kidney immortalized cell line as described in claim 1 in the study of drug nephrotoxicity assessment.

4. The application of the human normal kidney immortalized cell line as described in claim 1 in bioartificial kidney and cell therapy.

5. The application of the human normal kidney immortalized cell line as described in claim 1 in the study of kidney physiological and pathological mechanisms.

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