CN117417879A - Mouse proximal tubular epithelial cell line mRPTEC-B8 - Google Patents

Abstract

The invention discloses a mouse proximal tubular epithelial cell line mRPTEC-B8. The cell line mRPTEC-B8 is preserved in China Center for Type Culture Collection (CCTCC) at the year 7 and the month 18 of 2023, and the preservation number is CCTCC NO: C2023195. the cell line mRPTEC-B8 has morphological characteristics of mouse proximal tubular epithelial cells, expresses the proximal tubular epithelial cell markers CK18 and Lrp2, expresses the epithelial cell marker E-Cadherin, expresses the mesenchymal cell marker N-Cadherin in a low-level manner, and does not express the distal tubular cell marker uromodulin Umod and the collecting tube cell marker aquaporin Aqp2. The mRPTEC-B8 cell has the function of phagocytizing exogenous protein, and can be used for protein absorption experiments; but also for epithelial-to-mesenchymal transition (EMT) studies.

Description

Mouse proximal tubular epithelial cell line mRPTEC-B8

Technical Field

The invention relates to a mouse proximal tubular epithelial cell line mRPTEC-B8, belonging to the technical field of cell lines.

Background

Tubular epithelial cells of mammalian kidneys play an important role in the reabsorption of some nutrients, the removal of endogenous metabolic waste products, and the maintenance of water, electrolyte and acid-base balance. The tubular includes proximal tubular and distal tubular, and tubular epithelial cells account for about 90% of total cells of kidney, are the highest proportion of cell population in kidney, and are also injury targets sensitive to drugs such as cisplatin, folic acid, calcineurin inhibitors and hypoxia, and are important targets in China in kidney pathology and toxicology research. Researchers often separate and culture primary tubular epithelial cells for in vitro physiological, pathological, pharmacological, toxicological and other experimental researches, but the isolated and cultured tubular epithelial cells have larger heterogeneity due to factors such as experience differences of laboratory operators, different separation methods and the like, so that larger deviation is caused to experimental results.

To reduce experimental bias due to cell differences, researchers immortalized some tubular epithelial cell lines for in vitro studies, such as the mouse-derived proximal tubular epithelial cell line TKPTS, tubular epithelial cell lines TCMK-1, MRTEC, MTEC, etc.; a proximal tubular epithelial cell line HK-2 of human origin; rat tubular epithelial cell line NRK-52E, etc. Many laboratories use these cell lines for drug toxicology, epithelial-mesenchymal transition (EMT) and other studies in order to elucidate the mechanisms of pathological processes such as kidney injury and kidney fibrosis, which brings great convenience to scientific research.

Although these cell lines are widely used in various studies, these cell lines differ greatly in morphology, physiology, etc. from primary cultured tubular epithelial cells, such as morphology, most immortalized cells lose the round shape of primary cells, appear as spindle-shaped fibroblast-like cell morphology, and many tubular specific molecular markers disappear, such as aquaporin Aqp1, etc. critical for water uptake. Khundmri et al performed RNA sequencing of 14 proximal tubular epithelial cell lines from 6 species, and found that the transcriptome profile of none of the cells was perfectly matched to the primary proximal tubular epithelial cells. The gene expression profile was closest to that of a negative murine OK cell line, also expressing only 61 of 193 proximal tubular epithelial cell-specific expressed genes. However, because of the large difference between the genome of the negative mouse and the mouse and human, and the research of the cell line of the human or mouse is commonly performed by researchers, the research application of the OK cell line in the aspects of kidney physiology, pathology and the like is limited.

Protein reabsorption is one of the important functions of proximal tubular epithelial cells and is critical for recovery of protein components in blood. The protein uptake function of tubular epithelial cells depends on the heterodimeric receptor formed by the Lrp2 and Cubn proteins that they express. However, most immortalized proximal tubular epithelial cell lines no longer express the above genes, such as the widely used human proximal tubular epithelial cell line HK-2, and therefore these cell lines cannot be used for protein uptake studies, an important function of tubular cells, which researchers can only conduct by isolating primary tubular epithelial cells. For animals such as mice, kidney specimens can be obtained easily, primary tubular epithelial cells can be isolated and cultured, but some differences exist among cells extracted from different batches. In the case of humans, kidney specimens cannot be easily obtained to isolate primary cells, so this limits the study of human kidney protein absorption.

Thus, to date, no proximal tubular epithelial cell line has exhibited the function of all primary proximal tubular cells, each cell line being only available for a portion of specific physiological studies.

Disclosure of Invention

The purpose of the invention is that: in order to solve the problem that the existing mouse proximal tubular epithelial cell line cannot exhibit the functions of all primary tubular cells, the invention provides a mouse proximal tubular epithelial cell line mRPTEC-B8, and the mRPTEC-B8 cell line is close to the primary proximal tubular epithelial cells of the mouse in aspects of morphology, functions and the like.

In order to achieve the above purpose, the technical scheme adopted by the invention is to provide a mouse proximal tubular epithelial cell line mRPTEC-B8, wherein the mouse proximal tubular epithelial cell line mRPTEC-B8 is preserved in China Center for Type Culture Collection (CCTCC) for 7 months and 18 days in 2023, and the preservation number is CCTCC NO: C2023195.

preferably, the cell line mRPTEC-B8 expresses the tubular markers CK18 and Lrp2 protein, highly expresses the epithelial cell marker E-Cadherin, lowly expresses the mesenchymal cell marker N-Cadherin, and does not express the distal tubular cell marker uromodulin Umod and the collecting tube cell marker aquaporin Aqp2.

The invention also provides application of the mouse proximal tubular epithelial cell line mRPTEC-B8 in a protein absorption experiment.

The invention also provides application of the mouse proximal tubular epithelial cell line mRPTEC-B8 in the research of an epithelial-mesenchymal transition mechanism.

Compared with the existing mouse proximal tubular epithelial cell line, the invention has the beneficial effects that:

(1) The proximal tubular epithelial cell line mRPTEC-B8 has the morphological characteristics of the mouse proximal tubular epithelial cells, immortalized kidney cell B8 clones can form an epithelial cell-like single cell layer after being fully grown in a culture dish, and has a typical cobblestone-like morphology as that shown when primary proximal tubular cells are cultured, and when the cells are continuously cultured to reach higher cell density, a plurality of dome structures which are usually formed when the epithelial cells are cultured appear in the cell layer, so that the ion transport from the polarized top to the basement membrane direction is indicated. After a period of up to one year, the morphology of the cells was not significantly altered, indicating that they possessed a stable phenotype;

(2) The proximal tubular epithelial cell line mRPTEC-B8 of the invention expresses epithelial cell markers E-Cadherin and ZO-1 in a high mode and expresses mesenchymal cell markers N-Cadherin in a low mode; the mRPTEC-B8 cell expresses a protein absorption receptor Lrp2, has the function of phagocytizing exogenous proteins, and can be used for protein absorption experiments; the mRPTEC-B8 cells of the invention can also be used in epithelial-to-mesenchymal transition (EMT) studies;

(3) The near-end tubular epithelial cell line mRPTEC-B8 is not transfected with the large T antigen of SV40, so that the influence of the large T antigen on cells is reduced as much as possible, the cells do not carry resistance genes, and the screening of positive cells during the transfection of exogenous genes is facilitated.

Drawings

FIG. 1 is a morphology and growth curve of mRPTEC-B8 cells; a: the single cell layer formed by the mRPTEC-B8 cells presents a cobblestone-like morphology (left), and forms a fornix structure (right) at high cell density; b: growth curve of mRPTEC-B8 cells;

FIG. 2 is a molecular marker of mRPTEC-B8 expressing proximal tubular epithelial cells; a: western Blot detection of the results of various molecular markers; b: immunofluorescence results of CK 18; c: immunofluorescence results of ZO-1;

FIG. 3 is a graph showing the results of verification that mRPTEC-B8 cells can absorb foreign proteins; a: western Blot detects the expression of endocytosis and lysosomal associated proteins in mRPTEC-B8 and the other 4 tubular cell lines; b: mRPTEC-B8 and HK-2 endocytic BSA-FITC analysis;

FIG. 4 shows epithelial-to-mesenchymal transition (EMT) of mRPTEC-B8 upon TGF- β1 treatment; a: the mRPTEC-B8 cells are treated by TGF-beta 1 for 3 days, and then the expression changes of the fibrotic molecular markers Col1a1, alpha-Sma and the proximal tubular marker Lrp2 are analyzed by Western Blot; b: morphological changes of mRPTEC-B8 cells after 3 days of TGF-beta 1 treatment;

FIG. 5 shows STR genotype test results for mRPTEC-B8 cells.

Preservation description

The mouse proximal tubular epithelial cell line mRPTEC-B8 is preserved in China Center for Type Culture Collection (CCTCC) at the year 2023, 7 and 18, and the preservation number is CCTCC NO: c2023195, the preservation address is university of Wuhan in Wuhan, china.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.

The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

Example 1 immortalization preparation of mouse proximal tubular epithelial cell line mRPTEC-B8

The cells are prepared by a natural immortalization method. Taking an adult Kit-Creer; the kidney of Rosa26-LSL-tdTomato mice is prepared into a kidney single cell suspension by using a Multi Tissue Dissociation Kit kit of Meitian-Tsaoko company according to an operation procedure provided by the kit, and part of cells are inoculated into a culture dish for culture, wherein the culture medium is DMEM culture medium plus 10% fetal bovine serum (vitamin forest company), penicillin/streptomycin is added to prevent bacterial pollution, and the culture conditions are 37 ℃,5% carbon dioxide and saturated humidity. Pancreatin digests after every 3-7 days after cells are full of growth, approximately 1:4 passages, avoiding too rare cell densities after passage. Cells spontaneously immortalize after about two months of serial passages, during which the SV40 large T antigen isogene is not transfected.

Cell monoclonalization: immortalized cell populations were monocloned using limiting dilution. Cells were counted after pancreatin digestion, about 100 cells were diluted to 20mL of complete medium, inoculated into 96-well plates, 200 μl/well, and medium was changed every 3 days. During this period, the growth of cells in each well was observed daily with a microscope and wells containing individual clones were labeled. After about two weeks, single cell clones may form in a portion of the wells. And then, digesting clones formed by single cells by using pancreatin, transferring the clones into a new culture flask for culture and expansion, and collecting cells for freezing. The clone labeled B8 is mostly round, and is similar to the primary cultured mouse proximal tubular epithelial cells, so that the monoclonal cell clone is biologically preserved, the culture name is the mouse proximal tubular epithelial cell line mRPTEC-B8, and the clone is preserved in China Center for Type Culture Collection (CCTCC) at 7 months 18 of 2023, and the preservation number is CCTCC NO: C2023195.

example 2 morphological characterization and biological characterization of the mouse proximal tubular epithelial cell line mRPTEC-B8

1) The immortalized cell line mRPTEC-B8 prepared by the invention has the morphological characteristics of the epithelial cell intrinsic of the proximal renal tubule of the mouse. Immortalized kidney cell B8 clones, when grown in culture dishes, form an epithelial cell-like single cell layer with a typical cobblestone-like morphology as exhibited by primary proximal tubular cell cultures (left in fig. 1A), and when continued to culture cells to higher cell densities, multiple dome structures appear in the cell layer (right in fig. 1A), typically formed upon epithelial cell culture, suggesting the presence of polarized apical to basement membrane directed ion transport. The morphology of the cells was not significantly altered after cultivation for up to one year under standard cultivation conditions, indicating that they possessed a stable phenotype. Because the cells are proximal to the cultured primary miceThe tubular epithelial cells have similar morphological characteristics, so the cell line is named mRPTEC-B8 #mouse Renal Proximal Tubular Epithelial Cell-B8)

The doubling time for proliferation of the cell line was then examined. Taking 1×10 ⁵ Individual cells were seeded in 6-well plates, cells were digested and collected daily, counted, and continuously examined for 3 days, and the results were plotted as a growth curve (fig. 1B). The results show that the number of the cells is exponentially long, and the multiplication time of the cells is about 15 hours, so that the proliferation speed of the cells is relatively rapid.

2) The mRPTEC-B8 cells express a molecular marker of the proximal tubular cells. Typically isolated primary proximal tubular cells of mice can be immunofluorescently labeled with keratin 18 (CK 18) antibodies to detect their purity. We first examined the expression of CK18 in this cell using Western Blot, and the results indicate that this cell expresses CK18, and only human proximal tubular cells HK-2 express CK18 among the other four tubular epithelial cells MRTEC, MTEC, TCMK-1 and HK-2 (FIG. 2A). In addition, CK18 antibody immunofluorescence results also showed the presence of a fibrillar network structure within the mrtec-B8 cells, suggesting CK18 positivity (fig. 2B). The CK18 assay indicated that the cells were proximal tubular cells.

Protein reabsorption is an important function of proximal tubular cells, and heterodimers formed by Lrp2/Meglain and Cubn are receptors for protein absorption. To determine whether mRPTEC-B8 cells express a protein reabsorption related gene, the expression of Lrp2/Meglain in the cells was determined by Western Blot. The results showed that mRPTEC-B8 cells expressed Lrp2 protein, but none of the four tubular epithelial cells examined simultaneously MRTEC, MTEC, TCMK-1 and HK-2 expressed Lrp2 (FIG. 2A), although at lower levels compared to the kidney of the mice, suggesting that the cells may have proximal tubular-specific protein uptake functions. In addition, zonulin ZO-1 antibody staining showed that the mrtec-B8 cell contacts could form a tight junction structure (fig. 2C). These results further confirm that the cells are proximal tubular epithelial cells.

Water absorption is another important function of the proximal tubular cells to maintain the homeostatic balance of water in the internal environment. Aquaporin Aqp1 specifically expressed by proximal tubular cells was critical for water reabsorption, but Western Blot showed that mrtec-B8 cells, like the other four cells, did not express Aqp1 (fig. 2A), nor was Aqp1 detected in most tubular cell lines as reported in the literature. This result indicates that mRPTEC-B8 cells cannot be used in water transport studies.

Primary epithelial cells typically express the epithelial cell marker E-Cadherin in high levels and the mesenchymal cell marker N-Cadherin in low levels, but the expression of these two proteins is reversed in most immortalized epithelial cell lines. To detect the expression of these two markers in mRPTEC-B8 cells, we performed a Western Blot analysis, which showed that the epithelial cell marker E-Cadherin was expressed in mRPTEC-B8 cells, but not in the four tubular epithelial cells MRTEC, MTEC, TCMK-1 and HK-2; mRPTEC-B8 cells expressed a small amount of the interstitial cell marker N-Cadherin, and MTEC and HK-2 cells were similar thereto, but MRTEC and TCMK-1 cells highly expressed the interstitial cell marker N-Cadherin (FIG. 2A), indicating that the cells retained the molecular properties of the epithelial cells.

In addition, western Blot showed that mRPTEC-B8 cells did not express the distal tubular cell marker uromodulin Umod and the collecting tube cell marker aquaporin Aqp2 (FIG. 2A).

3) The mRPTEC-B8 cells can be used in protein absorption experiments. The mRPTEC-B8 cell expresses a protein absorption receptor Lrp2, which suggests that the cell has a protein absorption function. To verify this assumption, we first examined the protein uptake and the expression of lysosomal degradation-associated proteins in this cell. Western Blot results showed that mRPTEC-B8 cells did not express the heterodimer partner of Lrp2, cubn, but expressed the early endocytosis protein Rab5, the late endocytosis protein Rab7, the lysosomal proteins Lamp1 and CtsD, and the circulating endocytosis protein Rab11 (FIG. 3A), suggesting that the cells may have protein absorption functions. The expression of the above proteins in human proximal tubular cells HK-2 was similar to that in mRPTEC-B8 cells, with only a few of them being highly expressed in other cell lines (FIG. 3A).

To further verify whether mRPTEC-B8 cells have protein absorption functionProtein absorption experiments were performed, and the protein absorption experiment method was as follows: a round cell ultrathin glass climbing sheet is placed in a 12-hole plate and is connected with about 1 multiplied by 10 ⁵ The following day the cells were aspirated, complete medium containing 200. Mu.g/mL BSA-FITC was added, incubated at 4℃for 1 hour, and then incubated at 37℃for 30 minutes. The medium was aspirated, the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde, at room temperature for 20 min, protected from light. Cells were then washed with PBS, 10 min, 3 times, and nuclei were stained with DAPI. The cell slide was inverted and covered with anti-quencher slide glass, the periphery of the cell slide was blocked with nail polish, and the condition of endocytosis of BSA-FITC was detected with confocal microscope and images were collected.

The results showed that a dot-like green signal was visible in the mRPTEC-B8 cells (FIG. 3B), suggesting that BSA-FITC can be endocytosed into the cells, indicating that the cells have the function of absorbing proteins. In contrast, although HK-2 cells expressed most of the endocytosis-related protein, BSA-FITC was not absorbed due to the absence of Lrp2 expression (FIG. 3B, bottom). The result shows that the mRPTEC-B8 cells have the function of phagocytizing exogenous proteins and can be used for protein absorption experiments.

4) The mRPTEC-B8 cells can be used in epithelial-mesenchymal transition (EMT) studies. EMT experimental study method: about 2X 10 ⁵ Cells were inoculated into 6-well plates, TGF-. Beta.1 (10 ng/mL) was added, and cell morphology was changed in 1-3 days. The cells were collected, total protein was extracted, and the fibrosis markers Col1a1 and α -Sma were detected by Western Blot.

Failure to repair acute kidney injury in time can lead to kidney fibrosis, and transformation of tubular epithelial cells to interstitial cells (EMT) is an important aspect of the kidney fibrosis process, and HK-2 and TCMK-1 cells are often used for EMT mechanism research of human or mouse tubular epithelial cells. To verify whether mRPTEC-B8 cells could be used in EMT studies, the cells were tested for their molecular markers of differentiation into myofibroblasts 3 days after treatment with TGF- β1 in vitro. Western Blot results showed that mRPTEC-B8 cells were significantly increased in Col1a1 and α -Sma expression following TGF- β1 treatment, while the proximal tubular cell marker Lrp2 expression was decreased (FIG. 4A), indicating that the cells had differentiated towards myofibroblasts with loss of proximal tubular function. In addition, morphological observation of cells also revealed that the cells transformed from round to spindle-shaped fibroblast morphology after TGF- β1 treatment (fig. 4B). The above results demonstrate that mRPTEC-B8 cells are a better EMT study model.

5) And (5) detecting STR genotype of the mRPTEC-B8 cells. To determine the genotype of the immortalized cell line, cell samples were sent to Shanghai wing and applied biotechnology Co. The result shows that the cell line DNA typing does not find a matched cell line in the cell line search, and the cell line does not find multiallelic genes, has no cross contamination and no human pollution. For 9 mouse gene loci: 4-2, 5-5, 6-4, 6-7, 9-2, 12-1, 15-3, 18-3, X-1 (containing sex gene Amelogenin) and gene loci of 1 person: the results of STR analysis of D4S2408 (for detecting whether there is human contamination in the cells) are shown in fig. 5.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be limiting in any way and in nature, and it should be noted that several modifications and additions may be made to those skilled in the art without departing from the invention, which modifications and additions are also intended to be construed as within the scope of the invention.

Claims (4)

1. A mouse proximal tubular epithelial cell line mstec-B8, wherein said mouse proximal tubular epithelial cell line mstec-B8 was preserved in chinese typical culture collection (CCTCC) at day 7 and 18 of 2023 under the preservation number CCTCC NO: C2023195.

2. the mouse proximal tubular epithelial cell line mrptc-B8 of claim 1, wherein said cell line mrptc-B8 expresses proximal tubular epithelial cell markers CK18 and Lrp2 protein, expresses epithelial cell marker E-cadherein, low expresses mesenchymal cell marker N-cadherein, does not express distal tubular cell marker uromodulin Umod and manifold cell marker aquaporin Aqp2.

3. Use of the mouse proximal tubular epithelial cell line mrtec-B8 according to claim 1 or 2 in a protein uptake assay.

4. Use of the mouse proximal tubular epithelial cell line mrtec-B8 according to claim 1 or 2 in the study of epithelial-mesenchymal transition mechanism.