WO2023217126A1

WO2023217126A1 - Renal epithelial precursor-like cell, and preparation method therefor, preparation thereof and use thereof

Info

Publication number: WO2023217126A1
Application number: PCT/CN2023/092959
Authority: WO
Inventors: 张琴; 周伸奥
Original assignee: 上海赛立维生物科技有限公司
Priority date: 2022-05-10
Filing date: 2023-05-09
Publication date: 2023-11-16
Also published as: CN117018033A; CN117025506A; CN117025504A; WO2023217121A1; CN117018032A; WO2023217134A1; CN117025503A; CN117025502A; WO2023217132A1; WO2023217130A1; WO2023217123A1; WO2023217129A1; WO2023217128A1; CN117025508A; CN117025505A; WO2023217125A1; CN117025507A

Abstract

Provided in the present invention is a method for preparing a renal epithelial precursor-like cell, comprising the following steps: providing primary renal cells, placing the primary renal cells into a reprogramming medium for dedifferentiation culture until the confluence of the renal epithelial precursor-like cells is not less than 80%, and performing a digestion treatment on the renal epithelial precursor-like cells using a trypsin solution to obtain the renal epithelial precursor-like cells, wherein the reprogramming medium comprises a basic medium, hydrocortisone, cholera toxin, insulin, adenine, transferrin, and triiodothyronine. The present invention solves the problem of the difficulties of in-vitro proliferation of renal epithelial precursor-like cells during preparation in the prior art.

Description

Renal epithelial precursor-like cells and preparation methods, preparations and applications thereof

This application requires the priority of a Chinese patent application with a filing date of May 10, 2022, an application number of 2022105036786, and an invention title of "Biological preparations, cell derivatives, preparation methods and applications". The contents of the above application are incorporated herein by reference.

Technical field

The present invention relates to the field of biotechnology, and in particular to renal epithelial precursor-like cells and their preparation methods, preparations and applications.

Background technique

End-stage renal disease is the result of the continuous development of acute and chronic kidney disease. Traditional drug treatment and dialysis treatment are difficult to reverse the damage to the kidney structure and the continuous decline of renal function. In the end, it will inevitably progress to end-stage renal disease. The basic unit of kidney function is the nephron, which mainly includes glomeruli and renal tubules. Studies have shown that adult kidneys cannot completely regenerate nephrons, but the renal tubular epithelium has the potential to repair and regenerate damage. Studies have shown the presence of precursor cells in the human adult nephron, including epithelial precursor cells expressing CD133+ and CD24+ located in the glomerular capsule and scattered in the renal tubules. A mouse model based on lineage tracing of renal tubular epithelial cells found that terminally differentiated renal tubular epithelial cells can dedifferentiate into SOX9+-expressing precursor cells when the kidney is injured, proving that RTCs have cell fate plasticity. Studies have shown that it is possible to directly extract renal epithelial precursor cells from human urine, but due to their low content, isolation and extraction from urine requires tedious steps and is less efficient.

Urine is a reliable source of renal epithelial cells, and renal epithelial cells are obtained from urine and It has the advantage of being non-invasive and safe for renal epithelial cell regeneration treatment. Terminal differentiated renal epithelial cells have fate plasticity, and the use of renal epithelial precursor cells to alleviate acute and chronic kidney injury is a new therapy with great potential. However, isolation of adult renal epithelial precursor cells from urine currently requires tedious culture. The steps include the use of feeder cells and monoclonal screening. The sources of in vitro culture of adult kidney epithelial precursor cells include adult kidney tissue sorting and embryonic stem cells (English name: Embryonic stem cells, referred to as ESCs) or induced pluripotency. Stem cells (English name: Induced pluripotent stem cells, abbreviated as iPSCs) are differentiated. However, no matter which method is used, there are problems such as inefficiency, cumbersome steps, poor safety, and difficulty in cell expansion.

Therefore, it is necessary to provide a renal epithelial precursor-like cell and its preparation method, preparation and application to solve the above problems existing in the prior art.

Contents of the invention

The purpose of the present invention is to provide renal epithelial precursor-like cells and their preparation methods, preparations and applications, so as to solve the problem of difficulty in in vitro expansion of the preparation of renal epithelial precursor-like cells in the prior art.

In order to achieve the above objectives, the preparation method of renal epithelial precursor-like cells of the present invention includes the following steps:

S0: Provide renal primary cells;

S1: Put the renal primary cells into reprogramming medium for dedifferentiation culture until the confluence of the renal epithelial precursor-like cells is not less than 80%, use trypsin digestion solution to perform dedifferentiation culture on the renal epithelial precursor cells. Somatic-like cells are digested to obtain renal epithelial precursor-like cells, wherein, The reprogramming medium includes basal medium, hydrocortisone, choleramycin, insulin, adenine, transferrin and triiodothyronine.

The beneficial effect of the preparation method of renal precursor-like cells of the present invention is that: the renal primary cells are put into a reprogramming medium for dedifferentiation culture, and the reprogramming medium includes a basal medium, hydrocortisone , choleramycin, insulin, adenine, transferrin and agonists, so that the renal primary cells continuously realize self-renewal and proliferation, thereby the present application solves the problem of the preparation of renal epithelial precursor-like cells in the prior art. There is a problem of difficulty in amplification in vitro.

Preferably, the preparation method of the renal epithelial precursor-like cells further includes the following steps: S2: Expanding and culturing the renal epithelial precursor-like cells in the reprogramming medium until the renal epithelial precursor-like cells After the fusion degree of the sample cells is not less than 80%, the renal epithelial precursor-like cells are digested using the trypsin digestion solution to obtain passaged renal epithelial precursor-like cells.

Preferably, the reprogramming medium also includes growth factors, ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF-β signaling inhibitors, nutritional supplements and buffers.

Preferably, the content of hydrocortisone is 0.3-0.5 micrograms/ml based on the volume of the basal culture medium, and the content of choleramycin is 0.5×10 ^-10 -1.5×10 ^-10 M , the content of insulin is 4.5-5.5 ng/ml, the content of adenine is 1.3×10 ^-4 -2.3×10 ^-4 M, the content of transferrin is 4.5-5.5 μg/ml, The content of triiodothyronine is 1.5×10 ^-9 -2.5×10 ^-9 M.

Preferably, the content of the growth factor based on the volume of the basal culture medium is 50-90 ng/ml, the content of the ROCK kinase inhibitor is 8-12 μM, the content of the Wnt signaling pathway agonist is 2-4 μM, and the content of the TGF-β signaling inhibitor is 0.5-1.5 μM , the content of the nutritional supplement is 0.5-1.5%, and the volume content of the buffer solution does not exceed 5%.

Preferably, the renal epithelial precursor-like cells positively express at least one of CD24, CD133, SOX9, and CD44.

The present invention also provides a renal epithelial precursor-like cell, which is prepared by the method for preparing renal epithelial precursor-like cells.

The beneficial effects of the renal epithelial precursor-like cells of the present invention are that the preparation method of the renal epithelial precursor-like cells is simple, and the obtained renal epithelial precursor-like cells can reduce the impact of renal damage on the human body.

The present invention also provides a renal epithelial precursor-like cell preparation, which includes the renal epithelial precursor-like cells and a pharmaceutically acceptable carrier.

The beneficial effect of the renal epithelial precursor-like cell preparation of the present invention is that the obtained renal epithelial precursor-like cell preparation can alleviate renal damage and renal interstitial fibrosis caused by renal ischemia-reperfusion.

Preferably, the pharmaceutically acceptable carrier includes any one of physiological saline and compound electrolyte injection.

Preferably, the renal epithelial precursor-like cell preparation is used to intervene in an in vivo animal model to examine the effect on renal injury.

Preferably, the animal model includes a unilateral mouse renal ischemia-reperfusion model.

Description of drawings

Figure 1 is a photographic schematic diagram of the morphology of fourth-generation renal epithelial precursor-like cells according to an embodiment of the present invention;

Figure 2 is a photographic schematic diagram of the morphology of tenth generation renal epithelial precursor-like cells according to an embodiment of the present invention;

Figure 3 is a photographic schematic diagram of the morphology of third-generation control renal epithelial precursor-like cells according to an embodiment of the present invention;

Figure 4 is a comparative curve chart of the expansion and culture of renal epithelial precursor-like cells by the reprogramming medium and the basal medium of the present invention;

Figure 5 is a schematic diagram of the gene expression marker CD133 of renal epithelial precursor-like cells according to an embodiment of the present invention;

Figure 6 is a schematic diagram of the gene expression marker SOX9 of renal epithelial precursor-like cells according to an embodiment of the present invention;

Figure 7 is a schematic diagram of the gene expression marker CD24 of renal epithelial precursor-like cells according to an embodiment of the present invention;

Figure 8 is a schematic diagram of the gene expression marker CD44 of renal epithelial precursor-like cells according to an embodiment of the present invention;

Figure 9 is a schematic diagram of serum creatinine content in the control group and the experimental group;

Figure 10 is a comparison of photos obtained after taking left kidney tissue from the control group and the experimental group and staining it with picro-Sirius Sirius red staining and Masson's trichrome staining.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention. Unless otherwise defined, technical or scientific terms used herein shall have their ordinary meaning understood by one of ordinary skill in the art to which this invention belongs. The use of "comprising" and similar words herein means that the elements or things appearing before the word include the elements or things listed after the word and their equivalents, without excluding other elements or things.

Cell therapy has been applied to a variety of human diseases. Various human diseases include: islet cell transplantation, autologous chondrocyte cartilage repair, B cell therapy for systemic lupus erythematosus, hematopoietic stem cell transplantation for cancer treatment, etc., through the treatment of renal epithelial precursors. The cells are cultured to prepare renal epithelial precursor-like cells, and then it is possible to use renal epithelial precursor-like cells or cell-secreted factors to repair the structure and function of diseased kidney tissue and organize the regeneration of renal epithelial precursor-like cells. New therapies will bring new benefits to kidney diseases. Here comes new hope.

Currently, the isolation of adult renal epithelial precursor-like cells from urine involves the use of feeder cells and monoclonal screening, which is cumbersome and increases the risk of contamination; while the isolation of renal epithelial precursor-like cells from adult tissues requires invasion of the human body. The steps for flow cytometry or immunomagnetic bead sorting are cumbersome, and their practical application is limited due to possible cell contamination; ESCs/iPSCs differentiation is also cumbersome, and its safety has been widely questioned.

Establish a new method for the preparation of urine-derived renal epithelial precursor-like cells, make better use of urine as a cell source, establish innovative cell replacement therapy methods, and achieve the treatment of acute kidney disease. new breakthroughs in treatment methods and efficacy. The present invention establishes a technical system for reprogramming exfoliated epithelium derived from urine into renal epithelial precursors. It does not require invasive human operations, feeder cells, monoclonal sorting or iPSC stage, and efficiently obtains positive expression of CD24, Renal epithelial precursor-like cells of any one of CD44, CD133 and SOX9, and the renal epithelial precursor-like cells obtained by the present invention can significantly alleviate ischemia-reperfusion injury in mouse kidneys. Therefore, urine-derived renal epithelial precursors Such cells can be used in the clinical treatment of acute kidney injury.

The invention provides a method for preparing renal epithelial precursor-like cells, which includes the following steps:

S0: Provide renal primary cells;

S1: Put the renal primary cells into reprogramming medium for dedifferentiation culture until the confluence of the renal epithelial precursor-like cells is not less than 80%, use trypsin digestion solution to perform dedifferentiation culture on the renal epithelial precursor cells. The somatic cells are digested to obtain renal epithelial precursor-like cells, wherein the reprogramming medium includes basal medium, hydrocortisone, choleramycin, insulin, adenine, transferrin and triiodothyron Orthosine.

Specifically, the renal primary cells are cultured for dedifferentiation by placing them in a reprogramming medium, which includes basal medium, hydrocortisone, choleramycin, insulin, adenine, and transferrin. Proteins and agonists are used to enable the renal primary cells to continuously realize self-renewal and proliferation, thereby solving the problem of difficulty in in vitro expansion of the preparation of renal epithelial precursor-like cells in the prior art.

In some embodiments of the present invention, the preparation method of the renal epithelial precursor-like cells further includes the following steps: S2: Expand and culture the renal epithelial precursor-like cells in the reprogramming medium until the renal epithelial precursor-like cells are After the confluence of epithelial precursor-like cells is not less than 80%, use all The pancreatic digestion liquid digests the renal epithelial precursor-like cells to obtain passaged renal epithelial precursor-like cells.

In some embodiments of the present invention, the reprogramming medium further includes growth factors, ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF-β signaling inhibitors, nutritional supplements and buffers.

In some specific embodiments of the present invention, the growth factors include epidermal growth factor and basic fibroblast growth factor, the epidermal growth factor is derived from nearshore organisms, and the basic fibroblast growth factor is derived from nearshore organisms; ROCK kinase inhibitor Y-27632 is derived from Taoshu Biotech, the Wnt signaling pathway agonist CHIR99021 is derived from Taoshu Biotech, the TGF-β signaling inhibitor A8301 is derived from Taoshu Biotech, and the nutritional supplements include N2 nutrition Supplements and B27 nutritional supplements, the N2 nutritional supplement is sourced from Thermo Fisher.

In some embodiments of the present invention, the content of hydrocortisone is 0.3-0.5 μg/ml, and the content of choleramycin is 0.5×10 ^-10 -1.5×10 based on the volume of the basal culture medium. ^-10 M, the content of insulin is 4.5-5.5 ng/ml, the content of adenine is 1.3×10 ^-4 -2.3×10 ^-4 M, and the content of transferrin is 4.5-5.5 micrograms /ml, the content of triiodothyronine is 1.5×10 ^-9 -2.5×10 ^-9 M.

In some embodiments of the present invention, based on the volume of the basal culture medium, the content of the growth factor is 50-90 ng/ml, the content of the ROCK kinase inhibitor is 8-12 μM, and the Wnt signaling pathway The content of the agonist is 2-4 μM, the content of the TGF-β signal inhibitor is 0.5-1.5 μM, and the content of the nutritional supplement is 0.5-1.5%, so The volume content of the buffer solution shall not exceed 5%.

In some specific embodiments of the present invention, the growth factors include epidermal growth factor and basic fibroblast growth factor, and the content of the epidermal growth factor is 10-30 ng/ml based on the volume of the basal culture medium, The content of the basic fibroblast growth factor is 40-60 ng/ml.

In some embodiments of the present invention, the renal epithelial precursor-like cells positively express at least one of CD24, CD133, SOX9, and CD44.

The present invention also provides a renal epithelial precursor-like cell, which is prepared by the method for preparing renal epithelial precursor-like cells. Specifically, the preparation method of the renal epithelial precursor-like cells is simple, and the obtained renal epithelial precursor-like cells can reduce the impact of renal injury on the human body.

The present invention also provides a renal epithelial precursor-like cell preparation, which includes the renal epithelial precursor-like cells and a pharmaceutically acceptable carrier. The obtained renal epithelial precursor-like cell preparation can alleviate renal injury and renal interstitial fibrosis caused by renal ischemia-reperfusion.

In some embodiments of the present invention, the pharmaceutically acceptable carrier includes any one of physiological saline and compound electrolyte injection.

In some embodiments of the present invention, the renal epithelial precursor-like cell preparation is used to investigate the effect on renal injury after intervening in an in vivo animal model.

In some embodiments of the present invention, the animal model includes a unilateral mouse renal ischemia-reperfusion model.

Example

1. Statement on the nature of the starting organization and the legality of the source:

Healthy adult urine was used as the starting material to obtain renal epithelial precursor-like cells that positively express CD24, CD133 and SOX9. Specifically, the healthy adult urine comes from healthy adult volunteers who are no more than 70 years old and have no infectious virus infection after medical examination. The volunteers are fully informed of the purpose of obtaining the urine samples and signed an informed consent form.

2. Obtaining primary human kidney cells

Use a centrifuge tube to collect healthy adult urine samples. After diluting it with the same amount of PBS as the urine sample, immediately put the centrifuge tube into a centrifuge for centrifugation. After the centrifugation process is completed, discard the supernatant in the centrifuge tube and collect the centrifuge tube. Human kidney primary cells in , the centrifugation speed is 400g, the centrifugation temperature is 4 degrees Celsius, and the centrifugation time is 10 minutes.

3. Obtaining renal epithelial precursor-like cells

The components of the reprogramming medium include: epithelial cell growth factor EGF with a content of 20 ng/ml based on the volume of the basal medium DMEM/F12 (from Wuhan Pronosai Life Technology Co., Ltd.) and 50 ng/ml. g/ml basic fibroblast growth factor bFGF, 1% nutritional supplement N2 (1X), 1% nutritional supplement B27 (1X), 10 μM ROCK kinase inhibitor Y-27632 , Wnt signaling pathway agonist CHIR99021 at 3 μM, TGF-β signaling inhibitor A8301 at 1 μM, cortisone at 0.4 μg/ml, choleramycin at 10 ^-10 M, and 5 ng/ml of insulin in 1.8×10 ^-4 M adenine in Transferrin at 5 μg/ml, triiodothyronine at 2 × 10 ^-9 M, and 10% FBS.

Figure 1 is a schematic photographic diagram of the morphology of fourth-generation renal epithelial precursor-like cells according to an embodiment of the present invention; Figure 2 is a schematic photographic diagram of the morphology of tenth-generation renal epithelial precursor-like cells according to an embodiment of the present invention; Figure 3 is a schematic diagram of the implementation of the present invention. Photographic representation of the morphology of third-generation control kidney epithelial precursor-like cells from an example.

Place renal primary cells in a 6-well plate at a seeding area of 10,000 cells/cm², and add 2 ml of reprogramming medium to each well for dedifferentiation culture until the confluence of renal epithelial precursor-like cells is no less than 80%. After that, use trypsin digestion solution for digestion. The digestion time is 5 minutes. The renal epithelial precursor-like cells will continue to be expanded and cultured using reprogramming medium until the fusion degree of the renal epithelial precursor-like cells is not low. After reaching 80%, use trypsin digestion solution for digestion for 5 minutes to obtain the first generation renal epithelial precursor-like cells (denoted as P1), and cycle amplification and culture to obtain the fourth generation renal epithelial precursor cells. Epithelial precursor-like cells, the schematic diagram of the morphology of fourth-generation renal epithelial precursor-like cells (denoted as P4) is shown in Figure 1; continue to use reprogramming medium for expansion and culture to the tenth generation, and the tenth-generation renal epithelial precursor-like cells The schematic diagram of cell (denoted as P10) morphology is shown in Figure 2 .

Place primary renal cells in a 6-well plate at a seeding area of 10,000 cells/cm2, add 2 ml of basic medium to each well for dedifferentiation culture to obtain control renal epithelial precursor-like cells until the control renal epithelial precursor-like cells are obtained. After the confluence of the cells is not less than 80%, use trypsin digestion solution for 5 minutes. The control kidney epithelial precursor cells will continue to be expanded and cultured using basal medium until the control kidney epithelial precursor cells are expanded and cultured. After the confluence of epithelial precursor-like cells is no less than 80%, use trypsin digestion solution for digestion. The treatment time is 5 minutes to obtain the first-generation control renal epithelial precursor-like cells, and cycle expansion culture is performed to obtain the third-generation control human epithelial renal precursor-like cells. Refer to Figure 3. The use of basal culture medium can only Culture to fifth generation renal epithelial precursor-like cells.

Referring to Figures 1, 2 and 3, Figures 1 and 2 show that renal epithelial precursor-like cells cultured using reprogramming medium still have epithelial cell-like morphology even after being cultured to the tenth passage, indicating that the reprogramming medium supports renal epithelium. Long-term expansion of precursor-like cells; Figure 3 shows that renal epithelial precursor-like cells cultured in basal medium have become senescent by the third passage, indicating that other components other than the basal medium have an impact on renal epithelial precursor-like cells. The continued expansion of cells is indispensable.

Renal epithelial precursor-like cells are expanded and cultured in vitro using reprogramming medium, and can be expanded and cultured to at least the tenth passage. The proliferation capacity of renal epithelial precursor-like cells in each passage is shown in Table 1.

Table 1 Proliferative ability of renal epithelial precursor-like cells

From Table 1, it can be seen that the proliferation ability of renal epithelial precursor-like cells shows a trend of first increasing and then decreasing with the increase of generations. In the first generation, the proliferation ability of renal epithelial precursor-like cells is the largest. The proliferation ability of epithelial precursor-like cells is slightly higher than that of ninth-generation renal epithelial precursor-like cells.

Figure 4 is a comparative graph of the expansion and culture of renal epithelial precursor-like cells using the reprogramming medium and the basic medium of the present invention.

Referring to Figure 4, the abscissa represents the number of cell generations, and the ordinate represents the total number of cells. As can be seen from Figure 4, renal epithelial precursor-like cells can be expanded and cultured in vitro for at least ten generations using reprogramming medium, and renal epithelial precursor cells The number of somatic cells increases exponentially with each generation. However, the use of basic medium to expand and culture renal epithelial precursor-like cells in vitro can only be cultured for up to five generations, and at the fifth generation, the number of renal epithelial precursor-like cells is basically zero. Compared with the basic medium, the reprogramming medium of the present invention can continuously expand and culture renal epithelial precursor-like cells in vitro, and the number of renal epithelial precursor-like cells increases exponentially.

4. Flow cytometric detection of renal epithelial precursor-like cells

Figure 5 is a schematic diagram of the gene expression marker CD133 of renal epithelial precursor-like cells according to an embodiment of the present invention; Figure 6 is a schematic diagram of the gene expression marker SOX9 of renal epithelial precursor-like cells according to an embodiment of the present invention; Figure 7 is a schematic diagram of the gene expression marker CD24 of renal epithelial precursor-like cells in an embodiment of the present invention; Figure 8 is a schematic diagram of the gene expression marker CD44 of renal epithelial precursor-like cells in an embodiment of the present invention.

After the renal epithelial precursor-like cells are separated from the reprogramming medium, the renal epithelial precursor-like cells are washed with sterile PBS buffer, and then the renal epithelial precursor-like cells are digested with trypsin digestion solution and then centrifuged. After centrifugation, collect the cell pellet; add 100 μl of staining buffer (from Thermo Fisher) to the cell pellet to resuspend the cells in the flow tube, then add 5 μl of the flow cytometry antibody to be tested and incubate for 20 Minutes later, resuspend each flow tube with 400 μl of staining buffer to obtain a cell suspension. Cell suspension was analyzed by flow cytometry.

Specific steps for flow cytometry detection: For P4 generation renal epithelial precursor-like cells, aspirate the reprogramming medium, rinse with 5 ml of sterile PBS buffer, and then add 2 ml of trypsin digestion solution to the culture dish for digestion to obtain the cells. mixture, place the cell mixture in a 15ml centrifuge tube, put the centrifuge tube into a centrifuge, and centrifuge at a speed of 200g for 5 minutes. Discard the supernatant of the centrifuged cell mixture to obtain the cell pellet. things.

Add 700 μL of staining buffer to the cell pellet to resuspend the cell pellet. Transfer the resuspended cell pellet to six 1.5 ml centrifuge tubes with a specification of 100 μL/tube. Add 3 μL of the flow cytometry antibody to be tested into the six 1.5 ml centrifuge tubes mentioned above, and mix by pipetting. Place the centrifuge tubes in a refrigerator at 2-8°C for 30 minutes. Add sterile PBS buffer to each centrifuge tube at a dose of 800 μl/tube. Then put the centrifuge tubes into a centrifuge and mix at 300 g. Centrifuge at high speed for 5 minutes. After centrifugation, discard the supernatant, add 400 μL of staining buffer to each centrifuge tube to resuspend the cell pellet, and then transfer it to a flow tube. The resuspended cell mixture will be used for flow cytometric detection of surface markers. .

The names of antibodies used in surface marker flow cytometric detection are: CD24, CD44, and CD133.

Among them, CD24 was purchased from abcam, the catalog number is ab290730; CD44 was purchased from abcam, the catalog number is ab254530; CD133 was purchased from BD Biosciences, the catalog number is 566596; the staining buffer was purchased from BD Biosciences, the catalog number is 554656, and the trypsin digestion solution was purchased from Yuanpei, The item number is S310JV.

Intracellular marker staining of P4 generation renal epithelial precursor-like cells:

For the P4 generation renal epithelial precursor-like cells, aspirate the reprogramming medium, rinse with 5 ml of sterile PBS buffer, and then add 2 ml of trypsin digestion solution to the culture dish for digestion to obtain a cell mixture, and place the cell mixture Put the centrifuge tube into a centrifuge in a 15 ml centrifuge tube, and perform centrifugation at a speed of 200 g for 5 minutes. Discard the supernatant of the centrifuged cell mixture to obtain a cell pellet.

Add 1 ml of fixed membrane solution to the cell pellet, place the centrifuge tube in a refrigerator at 2-8°C for 50 minutes, add 2 ml of sterile PBS buffer to the centrifuge tube, and rotate the centrifuge tube at 300g for 5 minutes. After centrifugation, add 500 μl of staining buffer to the centrifuge tube to resuspend. Transfer the resuspended cell pellet to four 1.5 ml centrifuge tubes, with a specification of 100 μL/tube. Add 5 μL of the flow cytometry antibody to be tested to each of the above four 1.5 ml centrifuge tubes, mix by pipetting, and place the centrifuge tubes in an incubator containing 5% CO2 at 37°C for 30 minutes. After the incubation, add sterile PBS buffer to each centrifuge tube at a dose of 800 μl/tube, then place the centrifuge tube into a centrifuge and centrifuge at a speed of 300g for 5 minutes. After centrifugation, discard the supernatant, add 400 μL of staining buffer to each centrifuge tube to resuspend the cell pellet, and then transfer it to a flow tube. The resuspended cell mixture is subjected to flow cytometry for intracellular markers. detection.

The name of the antibody used for flow cytometric detection of intracellular markers is: Sox9.

Sox9 was purchased from abcam, the product number is ab208427; the fixed transmembrane solution was purchased from BD Biosciences, the product number is 554714, and the trypsin digestion solution was purchased from Yuanpei, the product number is S310JV. Staining buffer was purchased from BD Biosciences, catalog number 554656. The flow cytometry results are shown in Figure 5, Figure 6, Figure 7 and Figure 8.

Referring to Figures 5, 6, 7 and 8, the positivity rate of the positive peak of marker CD133 is 70.5%, the positivity rate of the positive peak of marker SOX9 is 99.8%, and the positivity rate of the positive peak of marker CD24 is 77.1 %, the positive rate of the positive peak of the marker CD44 is 99.6%. The positive rate of the positive peak is greater than 70%, indicating that the renal epithelial precursor-like cells positively express the marker. Therefore, the renal epithelial precursor-like cells of the present invention positively express CD133. , SOX9, CD24 and CD44.

5. Preparation of renal epithelial precursor-like cell preparations:

Take the fourth-generation renal epithelial precursor-like cells out of the liquid nitrogen tank and place them on dry ice for transportation; immediately submerge the fourth-generation renal epithelial precursor-like cells in a 37 degrees Celsius water bath, shake gently, and wait until the fourth generation of renal epithelial precursor-like cells The renal epithelial precursor-like cells are completely melted and shaken from side to side. Be careful not to immerse the hose joint in the water bath. Remove the melted fourth-generation renal epithelial precursor-like cells with a pipette and add them to a 50 ml sterile centrifuge tube. , put the centrifuge tube into a centrifuge for centrifugation. The speed of the centrifuge is 200g and the centrifugation time is 5 minutes. After the centrifugation is completed, discard the supernatant and use 0.9% sodium chloride injection to dissolve the fourth generation The renal epithelial precursor-like cells are diluted to prepare a renal epithelial precursor-like cell preparation, and the renal epithelial precursor-like cell preparation is controlled to contain 2×10 ⁷ renal epithelial precursor-like cells per milliliter.

6. Application of renal epithelial precursor-like cells in animal models and demonstration of effects:

Cell therapy has been used in a variety of human diseases, including islet cell transplantation, autologous chondrocyte cartilage repair, B cell therapy for systemic lupus erythematosus, and hematopoietic stem cell transplantation. Implants to treat cancer, etc. Through the cultivation and application of renal epithelial precursor-like cells, new therapies that use cells or cell-secreted factors to repair the structure and function of diseased kidney tissue and regenerate tissue cells have brought new hope to kidney diseases.

Previous studies have demonstrated that exogenous infusion of renal epithelial precursor-like cells can alleviate various acute kidney injuries in mouse models. Intravenous infusion of renal epithelial precursor-like cells that positively express CD133 and CD24 can alleviate rhabdomyolysis-induced acute kidney injury in immunodeficient mice; similarly, renal epithelial precursor-like cells in doxorubicin-induced focal segments It can reduce proteinuria and improve glomerular damage in the mouse model of chronic glomerulosclerosis (FSGS).

The animal model refers to the unilateral mouse renal ischemia-reperfusion model.

The preparation method of the unilateral mouse renal ischemia-reperfusion model includes the following steps: select 8-9 year old C57BL/6 mice, and after the C57BL/6 mice are anesthetized by gas, perform an operation along the midline of the abdomen of the C57BL/6 mice. An incision of 1.5 to 2.0 cm was made, and the skin and peritoneum were separated layer by layer, and finally the abdominal cavity of the C57BL/6 mouse was entered. After the renal pedicle was found, the left and right renal pedicles were quickly blocked with non-invasive micro-arterial clamps. It can be seen that the kidneys of C57BL/6 mice are composed of The bright red color turned into purple-black. The blocking time of the non-injurious micro-arterial clamp was 22 minutes. Then the non-injurious micro-arterial clamp was removed. At the end of the operation, the incision was sutured to close the abdominal cavity to obtain a unilateral mouse renal ischemia-reperfusion model.

The renal epithelial precursor-like cell preparation was injected into the unilateral mouse renal ischemia-reperfusion model as the experimental group. The specific steps include: injecting the renal epithelial precursor-like cell preparation into part of the unilateral mouse renal ischemia-reperfusion model through the tail vein. , 1 × 10 ⁶ renal epithelial precursor-like cells were administered per mouse; the unilateral mouse renal ischemia-reperfusion model was injected with PBS buffer as a control group. The specific steps included: PBS buffer was injected through the tail vein. Injection into some unilateral mice lacking in the blood reperfusion model.

Figure 9 is a schematic diagram of the serum creatinine content in the control group and the experimental group; Figure 10 is a photo comparison of the left kidney tissue taken from the control group and the experimental group and stained with picro-Sirius Sirius red staining and Masson's trichrome staining, Figure 10 Middle scale bar is 100 μm.

In the unilateral mouse ischemia-reperfusion model in the experimental group and the control group, 14 days after injection, the mice were euthanized with carbon dioxide, and 5 ml of blood was extracted from the heart of the mice, and 5 ml of blood was placed in a procoagulation tube. , put the procoagulation tube into a centrifuge for centrifugation. The centrifugation speed is 3000 rpm and the centrifugation time is 10 minutes. After the centrifugation process, take the supernatant from the procoagulation tube and detect the experimental group and For the serum creatinine content in the control group, specifically refer to Figure 8; the left kidneys of the mice in the unilateral mouse ischemia-reperfusion model in the experimental group and the control group were cut into two halves along the coronal plane, and one half was fixed in volume. The left kidney was fixed in 10% formalin solution and stored at room temperature. It was fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. Then it was cut into 44m thick slices. The left kidney slices were routinely used. Picro-Sirius Sirius red staining and Masson's trichrome (MT) staining were used to stain collagen deposition. Please refer to Figure 9 for details.

Referring to Figure 9, the ordinate Serum Cr represents the content of serum creatinine in μmol/L. It can be seen from the figure that the serum creatinine in the experimental group of mice is lower than the serum creatinine in the control group of mice, indicating that by Treatment with the renal epithelial precursor-like cell preparation of the present invention alleviated the renal damage of the mice in the experimental group; referring to Figure 10, the mice in the control group had loss of proximal renal tubule structure, dilation of the distal renal tubule, and visible casts. Interstitial fibrosis occurred, and the renal tubular structure and interstitial fibrosis of the mice in the experimental group were significantly improved compared with the control group. It can be seen that the renal epithelial precursor-like cell preparation of the present invention can alleviate renal ischemia-reperfusion injury and renal Renal fibrosis caused by ischemia-reperfusion.

Establish an innovative method for preparing urine-derived renal epithelial precursor-like cells, make better use of urine as a cell source, establish innovative cell replacement therapy methods, and achieve new breakthroughs in the treatment and efficacy of acute kidney disease. We have established a technical system for reprogramming urine-derived exfoliated epithelium into renal epithelial precursor-like cells, which does not require invasive human operations, feeder cells, monoclonal sorting or iPSC stage, and can efficiently obtain positive expression of CD24 through chemical reprogramming methods. , CD44, CD133 and SOX9, can significantly alleviate renal ischemia-reperfusion injury in mice. Therefore, urine-derived renal epithelial precursor-like cells may be used in the treatment of clinical acute kidney injury.

Although the embodiments of the present invention have been described in detail above, it will be obvious to those skilled in the art that various modifications and changes can be made to these embodiments. However, it should be understood that such modifications and changes are within the scope and spirit of the invention as described in the claims. Furthermore, the invention described herein is capable of other embodiments and of being practiced or carried out in various ways.

Claims

A method for preparing renal epithelial precursor-like cells, characterized by comprising the following steps:

S0: Provide renal primary cells;

S1: Put the renal primary cells into reprogramming medium for dedifferentiation culture until the confluence of the renal epithelial precursor-like cells is not less than 80%, use trypsin digestion solution to perform dedifferentiation culture on the renal epithelial precursor cells. The somatic cells are digested to obtain renal epithelial precursor-like cells, wherein the reprogramming medium includes basal medium, hydrocortisone, choleramycin, insulin, adenine, transferrin and triiodothyron Orthosine.
The method for preparing renal epithelial precursor-like cells according to claim 1, further comprising the following steps:

S2: Expand and culture the renal epithelial precursor-like cells in the reprogramming medium until the confluence of the renal epithelial precursor-like cells is not less than 80%, and then use the trypsin digestion solution The renal epithelial precursor-like cells are digested to obtain passaged renal epithelial precursor-like cells.
The method for preparing renal epithelial precursor-like cells according to claim 2, wherein the reprogramming culture medium further includes growth factors, ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF-β signaling inhibitors, Nutritional supplements and buffers.
The method for preparing renal epithelial precursor-like cells according to claim 3, wherein the content of hydrocortisone is 0.3-0.5 μg/ml based on the volume of the basal culture medium, and the The content of choleramycin is 0.5×10 -10 -1.5×10 -10 M, The content of the insulin is 4.5-5.5 ng/ml, the content of adenine is 1.3×10 -4 -2.3×10 -4 M, and the content of transferrin is 4.5-5.5 μg/ml, so The content of triiodothyronine is 1.5×10 -9 -2.5×10 -9 M.
The preparation method of renal epithelial precursor-like cells according to claim 4, characterized in that, based on the volume of the basal culture medium, the content of the growth factor is 50-90 ng/ml, and the ROCK The content of the kinase inhibitor is 8-12 μM, the content of the Wnt signaling pathway agonist is 2-4 μM, the content of the TGF-β signaling inhibitor is 0.5-1.5 μM, and the content of the nutritional supplement is 0.5- 1.5%, and the volume content of the buffer solution does not exceed 5%.
The method for preparing renal epithelial precursor-like cells according to claim 1, wherein the renal epithelial precursor-like cells positively express at least one of CD24, CD133, SOX9, and CD44.
A renal epithelial precursor-like cell, characterized in that it is prepared by the preparation method of renal epithelial precursor-like cells according to any one of claims 1 to 6.
A renal epithelial precursor-like cell preparation, characterized by comprising the renal epithelial precursor-like cells according to claim 7 and a pharmaceutically acceptable carrier.
The renal epithelial precursor-like cell preparation according to claim 8, wherein the pharmaceutically acceptable carrier includes any one of physiological saline and compound electrolyte injection.
An application of a renal epithelial precursor-like cell preparation, characterized in that the renal epithelial precursor-like cell preparation as described in any one of claims 8-9 is used to intervene in an in vivo animal model The impact on renal damage was then examined.
The application of the renal epithelial precursor-like cell preparation according to claim 10, wherein the animal model includes a unilateral mouse renal ischemia-reperfusion model.