Human normal renal immortalized cell line NRC-X1 and its use

Abstract

The application belongs to the field of microorganism animal cell lines, and relates to a human normal kidney immortalized cell line NRC-X1 and application thereof. The human normal kidney immortalized cell line is named NRC-X1, and the preservation number is CCTCC NO: C2025168. The application of the human normal kidney immortalized cell line NRC-X1 in a virus infection mechanism. The application of the human normal kidney immortalized cell line NRC-X1 in research on drug nephrotoxicity evaluation. The application of the human normal kidney immortalized cell line NRC-X1 in a biological artificial kidney and cell therapy. The application of the human normal kidney immortalized cell line NRC-X1 in research on kidney physiological and pathological mechanisms.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial animal cell lines, and particularly relates to a human normal kidney immortalized cell line NRC-X1 and application thereof. BACKGROUND

[0002] The kidney is an important organ of the human body, and its basic function is to generate urine, thereby removing metabolic products and certain wastes and poisons in the body, while retaining water and other useful substances such as glucose, protein, amino acids, sodium ions, potassium ions, bicarbonate, etc. through reabsorption function, so as to regulate water and electrolyte balance and maintain acid-base balance. The kidney also has endocrine function, generating renin, erythropoietin, active vitamin D3, prostaglandin, kinin, etc., and is also the degradation site of part of endocrine hormones and the target organ of extrarenal hormones. These functions of the kidney ensure the stability of the internal environment of the body and enable normal metabolism to proceed.

[0003] The kidney has strong compensatory capacity, and when the kidney damage exceeds 75%, the residual kidney tissue can still maintain normal physiological functions of the human body through compensation for a period of time, and when the kidney continues to be damaged, metabolic waste in the human body cannot be excreted, directly threatening life.

[0004] Immortalized human normal kidney cells are a bridge connecting basic research and clinical application, and are irreplaceable, especially in drug development, toxicity testing and disease mechanism research. With the development of organoids and 3D culture technology, its application value will be further expanded.

[0005] The immortalized human kidney cell lines currently used in medical research are: HK-2, human kidney proximal tubular epithelial cells (SV40 immortalized), commonly used for toxicity research. RPTEC / TERT1, telomerase immortalized human kidney proximal tubular cells, retaining more characteristics close to primary cells. HEK293, human embryonic kidney cells (adenovirus transfection), widely used in molecular biology and virus packaging. However, to date, there is no literature report on Chinese normal kidney immortalized cell lines. Therefore, it is necessary to establish a Chinese normal kidney immortalized cell line. SUMMARY

[0006] The purpose of the present application is to provide a Chinese human normal kidney immortalized cell line NRC-X1 and its application in view of the current situation that there is no Chinese normal kidney immortalized cell line, thereby providing a research model for kidney medical research of the Chinese people.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] The present application provides a human normal kidney immortalized cell line named human normal kidney immortalized cell line NRC-X1, which has been preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: C2025168.

[0009] Establishment of cell line: The present application uses normal kidney tissue from a 70-year-old female patient's renal pelvis and kidney cancer surgical resection specimen, which is digested by collagenase type II and neutral protease mixture for primary culture, and successfully cultures a kidney-derived immortalized cell line through standard cell culture technology and SV40 large T antigen gene transfection technology, and is named NRC-X1.

[0010] The cell line has been preserved in China Center for Type Culture Collection (CCTCC for short) on May 29, 2025, and the address of the preservation center is: China. Wuhan. Wuhan University, and the preservation number of the preservation center is CCTCC NO: C2025168.

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

[0012] 1. Cell growth characteristics: cells grow adherently, and cells are mainly short spindle-shaped.

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

[0014] 3. Cells are confirmed to be new human cell lines by STR detection.

[0015] 4. The results of cell immunohistochemistry show that NRC-X1 is a kidney-derived cell line.

[0016] 5. Cells are immortalized by SV40 transfection, can transcribe SV40T mRNA and translate SV40T protein.

[0017] The application of the human normal kidney immortalized cell line NRC-X1 in the mechanism of viral infection.

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

[0019] The application of the human normal kidney immortalized cell line NRC-X1 in biological artificial kidney and cell therapy.

[0020] The application of the human normal kidney immortalized cell line NRC-X1 in the research 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. The normal kidney cell biological characteristics of the cell line can be used to simulate the physiological or pathological state of the kidney, and to study the mechanism of kidney disease, drug action target and efficacy evaluation.

[0022] The application of the human normal kidney immortalized cell line NRC-X1 in drug screening and toxicology research, by detecting the influence of drugs on cell proliferation, marker expression or function, to evaluate the renal toxicity or therapeutic effect of drugs, to provide an in vitro model for kidney-related drug development.

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

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

[0025] The application constructs a brand new Chinese human normal kidney immortalized cell line NRC-X1, which can be used as a cell model for drug development, renal toxicity testing, disease mechanism research and biological artificial kidney research. It provides a brand new tool for kidney disease research and helps to promote the research progress of Chinese kidney disease. BRIEF DESCRIPTION OF DRAWINGS

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

[0027] Figure 2 It is the HE staining result of NRC-X1 cells.

[0028] Figure 3 It is the immunohistochemical result of NRC-X1 cells. A represents the positive expression of CD10 in cells; B represents the positive expression of CK7 in cells; C represents the positive expression of PAX2 in cells; D represents the positive expression of E-cadherin in cells; and E represents the positive expression of Vimentin in cells.

[0029] Figure 4 It is the level of SV40T mRNA of NRC-X1 cells.

[0030] Figure 5 It is an indirect immunofluorescence image of NRC-X1 and primary cells. It can be detected that NRC-X1 cells express SV40T protein, while the primary cell control group does not express SV40T protein, and the result identifies NRC-X1 at the protein level.

[0031] Figure 6 It is the growth curve of NRC-X1 cells. The abscissa is the cell culture time, and the ordinate is the cell number. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present application more clear, the following examples will further illustrate the present application with reference to the drawings. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application. On the contrary, the present application covers any substitution, modification, equivalent method and scheme defined by the claims within the essence and scope of the present application.

[0033] The experimental methods described in the following examples are all conventional methods unless otherwise specified.

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

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

[0036] The normal renal tissue surgically removed from a patient was subjected to primary culture after mixed enzyme digestion, and a normal renal immortalized cell line capable of continuous passage was successfully established, which has been passed to the 40th generation and the cell characteristics remain stable.

[0037] The present application uses a surgically removed specimen from a 70-year-old female patient, which is subjected to primary culture after mixed digestion with collagenase type II and neutral protease, and a normal renal immortalized cell line is cultured and established by cell culture technology and viral transfection technology, named NRC-X1. It was preserved on May 29, 2025, and the preservation unit is China Typical Culture Collection Center (CCTCC, address: Wuhan University, China Typical Culture Collection Center, Wuhan, China), preservation center preservation number: CCTCC NO: C2025168.

[0038] II. Biological property detection of human normal renal immortalized cell line NRC-X1

[0039] 1. Cell morphological observation

[0040] The cells were observed after stable growth and passage. The results showed that the cells were arranged in an epithelial-like manner under a microscope, and the cells grew adherently, with short spindle-shaped cells as the main cells. The morphological observation of NRC-X1 cells under a microscope is shown in Figure 1 . Figure 1 Figure A is the cell morphology under low magnification (40x), Figure 1 Figure B is 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. After 48 h, the medium was removed, the glass slides were washed twice with PBS, fixed with 4% paraformaldehyde solution for 15 min, and then air-dried in a ventilated place. After air-drying, the glass slides were permeabilized with 0.5% Triton X-100 solution for 20 min, and then subjected to HE staining. The results are shown in Figure 2 .

[0043] Immunohistochemical staining: After deparaffination and rehydration, the glass slides were immersed in a 10 mM sodium citrate solution at pH 6.0, boiled for 90 s, and then incubated in a 3% hydrogen peroxide solution at 37°C for 15 min. Then, 100 μL of normal goat serum was added, and the glass slides were incubated in a thermostat at 37°C for 15 min to reduce non-specific antibody binding and background staining. Then, the glass slides were incubated with anti-CD10, anti-CK7, anti-PAX2, anti-E-cadherin, and anti-Vimentin at 37°C for 12 h, respectively. Then, secondary antibodies were added, and the glass slides were incubated at room temperature for 50 min. Color development was performed using a DAB staining kit. After the glass slides were washed with running water for 5 min, they were subjected to hematoxylin re-staining, gradient ethanol dehydration, xylene transparency, neutral resin mounting, and then observed under an inverted microscope (Olympus, IX73+DP74). The results are shown in Figure 3 .

[0044] Figure 3 A in FIG. 1 indicates that CD10 is positively expressed in the cells, and the staining is located in the cytoplasm or cell membrane. Figure 3 B in FIG. 1 indicates that CK7 is positively expressed in the cells, and the staining is located in the cytoplasm. Figure 3 C in FIG. 1 indicates that PAX2 is positively expressed in the cells, and the staining is located in the nucleus. Figure 3 D in FIG. 1 indicates that E-cadherin is positively expressed in the cells, and the staining is located in the cytoplasm or cell membrane. Figure 3 E in FIG. 1 indicates that Vimentin is positively expressed in the cells, and the staining is located in the cytoplasm.

[0045] 3. Identification of cell expression of SV40 gene

[0046] Total RNA was extracted from the newly isolated primary cells (amplified for 5 days) and NRC-X1 cells passed to the 35th generation, respectively, and the RNA was reversely transcribed into cDNA. Real time quantitative polymerase chain reaction (RTqPCR) was used to detect the mRNA expression level of the SV40 target gene of the primary and NRC-X1 cells, GAPDH was used as an internal reference, the primer sequence is shown in Table 1, and the dye method (TB Green) was used to perform Real-time fluorescence quantitative qPCR detection using Mastercycler nexus GSX1 gradient PCR instrument (Eppendorf, Germany). The qPCR reaction system was as follows: DNA template 100 ng, 2 x TB Green Premix Ex Taq II Fast qPCR 12.5 μL, 10 Upstream primer 1 μL, 10 Downstream primer 1 μL, sterilized water supplemented 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, annealing at 60 °C for 30 s, a total of 40 cycles. Relative quantitative analysis was performed according to the method, and the results are shown in Figure 4 . Figure 4 The column chart of the relative level of SV40T mRNA in NRC-X0 (primary cells) and NRC-X1 cells. The horizontal coordinate is the cell type, which is NRC-X0 and NRC-X1 (35th generation cells) respectively; the vertical coordinate is the relative level of SV40T mRNA. The black column in the figure represents NRC-X0, and the gray column represents NRC-X1. It can be seen that compared with the primary cells, the SV40T mRNA level of NRC-X1 is much higher than that of NRC-X0, and the error line at the top of the column reflects the degree of data dispersion. The upper "** Figure 4 " mark indicates that the difference between the two groups is extremely significant (P < 0.0001), which indicates that the SV40 gene is successfully integrated into NRC-X1 cells and effectively transcribed into mRNA from the gene transcription level, which proves that the cell immortalization is successful from the gene level.

[0047] Table 1 Primer sequence of 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 in logarithmic growth phase (amplified for 5 days) and NRC-X1 cells passed to the 35th generation were inoculated in 12-well plates. When the cells grew to a monolayer, the culture medium was discarded, the cells were washed with PBS for 3 times, 4% paraformaldehyde was added for fixation at 4°C for 30 min, 0.5 mL of permeabilization agent 0.25% Triton X-100 was added, and the cells were incubated at room temperature for 10 min. The cells were washed with PBS containing 1% BSA for 3 times. After incubation with mouse anti-SV40 monoclonal antibody at room temperature for 2 h, the cells were washed with PBS containing 1% BSA for 3 times, 1:50 diluted fluorescently labeled goat anti-mouse IgG antibody and a DAPI solution with a final concentration of 5 μg / mL were added to each well, and the cells were incubated at room temperature in the dark for 2 h. The cells were washed with PBS containing 1% BSA for 3 times, 0.5 mL of PBS was added to each well, and the cells were observed under a fluorescence microscope and photographed. Three sets of replicates were set up to ensure the reproducibility of the results. The indirect immunofluorescence images of NRC-X1 and primary cells are shown in Figure 5 , in which A and C show the staining of cell nuclei with 4', 6-diamidino-2-phenylindole (DAPI), showing blue fluorescence, clearly showing the nuclear structure and distribution of the cells, which can be used as a reference for cell localization; B and D use mouse anti-SV40T mAb to detect SV40T protein, and the positive expression sites show green fluorescence. The results show that green fluorescence can be detected in NRC-X1 cells, indicating that the cells express SV40T protein, while the primary cell control group has no green fluorescence signal, indicating that the primary cells do not express SV40T protein, thereby identifying the immortalized characteristics of NRC-X1 cell line at the protein level. In combination with the results of DAPI staining and SV40T protein detection, the expression position of the target protein in the cell and the corresponding relationship of the overall structure of the cell are directly presented.

[0052] 5. STR identification

[0053] Logarithmic growth phase NRC-X1 cells were collected after trypsin digestion and sent to Suzhou Zhan Data Biotechnology Company for STR analysis to confirm that NRC-X1 cells are a completely new human cell line. The results are shown in Table 2.

[0054] Table 2 NRC-X1 cell STR profile report

[0055]

[0056] The STR typing results show that the STR spectrum of the NRC-X1 cell line is significantly different from the typical STR spectrum of known common kidney cell lines (such as HK-2, HEK293, RPTEC / TERT1), and no matching items are found in the public STR database (such as ExPASy), confirming that the NRC-X1 cell line is a brand new human cell line and is not cross-contaminated with the above-mentioned common cell lines.

[0057] 6. Determination of cell doubling time

[0058] Select NRC-X1 cells in the logarithmic growth phase, digest with 0.25% trypsin at 37°C for 5 min, add 3 times the volume of complete medium (a mixture of RPMI-1640 medium, 10% fetal bovine serum, 1% penicillin (100 U / mL)-streptomycin (100 ) solution) to the trypsin, centrifuge to collect the precipitate, resuspend the cells with an appropriate amount of complete medium to prepare a single cell suspension. Count the prepared single cell suspension, and inoculate 0, 2.5 x 10 3 , 4 x 10 3 , 7.5 x 10 3 , 1 x 10 4 , 1.25 x 10 4 , 1.5 x 10 4 , 1.75 x 10 4 and 2 x 10 4 cells in 96-well plates, 6 replicates per group, in a 37°C, 5% CO2 incubator. After the cells adhere, add CCK8 working solution 110 μL / well (RPMI-1640:CCK8=10:1), incubate for 2 h, and measure the absorbance at 450 nm. Plot the standard curve with cell number as the abscissa and OD value as the ordinate. Inoculate 5 x 10 3 cells in 96-well plates and set up a blank control, and measure the absorbance at 450 nm at 0, 24, 48, 72 and 96 h. Plot the growth curve of NRC-X1 cells using GraphPad Prism 8.0.2 with inoculation time as the abscissa and cell number as the ordinate, and calculate the cell doubling time. The cell doubling time is about 30 h. The growth curve of NRC-X1 cells is shown in Figure 6 . Figure 6 The growth trend of the cells at different time points is clearly shown, providing intuitive data support for a deep understanding of the proliferation characteristics of the cells.

[0059] The above examples are only preferred embodiments of the present application and cannot be considered as limiting the scope of the present application. Any equivalent changes and improvements made within the scope of the present application should still be included in the scope of the present application.

Claims

1. A human normal kidney immortalized cell line, characterized in that The cell line is named as human normal renal immortalized cell line NRC-X1, and the preservation number is CCTCC NO: C2025168.

2. The use of the human normal renal immortalized cell line according to claim 1 in the preparation of a cell model for drug development.

3. The use of the human normal renal immortalized cell line according to claim 1 in the preparation of a cell model for nephrotoxicity test.

4. The use of the human normal renal immortalized cell line according to claim 1 in the preparation of a cell model for disease mechanism research and bioartificial kidney research.

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