A method for preparing urine-derived renal progenitor cells and its application in diseases

Abstract

The present invention relates to a method for preparing urine-derived renal progenitor cells and their application in diseases. The prepared renal progenitor cells have stem cell characteristics and can be used to prepare cells for treating renal diseases caused by ischemia-reperfusion injury. The cells have good therapeutic application value.

Description

Technical Field

[0001] The present application relates to the biological field, and specifically to a method for preparing urine-derived renal progenitor cells and their application in diseases. Background Art

[0002] Progenitor cells belong to adult stem cells and are undifferentiated multipotent or specialized stem cells. Progenitor cells exist in various adult tissues of organisms and are responsible for the repair and regeneration process after tissue damage. After the damage occurs, progenitor cells can be mobilized and activated, proliferate in large numbers and migrate to the damaged site, differentiate into mature cells, and replace the damaged tissue. Corresponding progenitor cells have been identified in various tissues and organs of the human body, such as blood progenitor cells, skin progenitor cells, small intestinal progenitor cells, lung progenitor cells, etc. Because progenitor cells have the "magical" function of helping tissues and organs repair and regenerate, scientists are trying to cultivate progenitor cells and transplant them into patients to treat a variety of degenerative diseases.

[0003] Recent studies have found that renal progenitor cells (RPCs) are a type of stem cell-like cell with unlimited proliferation and multidirectional differentiation potential. They can play an important role in the repair of acute kidney injury (AKI) and ischemia-reperfusion injury (IRI), and have gradually attracted people's attention. The latest research has found that RPCs, as a type of cell with stem cell-like unlimited proliferation ability and multidirectional differentiation potential, can express stem cell markers (CD24, CD133), stem cell transcription factors (Oct-4, Pax-2) and proteins that respond to cell proliferation (PCNA, vimentin), etc. Under physiological conditions, the cells are in a dormant state. When renal function is impaired, the above cells can rapidly proliferate and differentiate and participate in the repair of renal injury.

[0004] A large number of studies support that RPCs originate from the kidney itself, but it cannot be denied that they may originate from the bone marrow or other adjacent tissues and organs. Bone marrow-derived stem cells can migrate to the injured area during the repair of AKI or IRI, participate in the proliferation and regeneration of renal tubular epithelial cells, and rebuild the normal physiological structure of the renal tubules, suggesting that RPCs may also originate from the bone marrow. Studies have found that in the repair of AKI and IRI, bone marrow-derived stem cells cannot differentiate into renal tubular epithelial cells, but differentiate into endothelial cells to promote local microcirculation in the injured area, indirectly participating in the reconstruction of renal tubular structure. Other studies have found that about 20%-50% of bone marrow-derived stem cells are transformed into renal tubular epithelial cells through cell fusion rather than dedifferentiation, participating in the repair of IRI and AKI. Studies on the mouse model of unilateral ureteral obstruction found that the total amount of BrdU in the renal tissue after obstruction did not change significantly, suggesting that there was no infiltration of BrdU+ cells from other tissues during this process, that is, the RPCs originated from the kidney itself, not extrarenal tissues. The study also found that no other cells or non-tubular cells were involved in the renal tubules before and after AKI, indicating that during the AKI repair process, the kidney's own RPCs proliferated, differentiated and formed renal tubular structures.

[0005] At present, the cell biological mechanism of RPCs developing from single cells into tubular structures is still unclear. Studies have found that the above process includes the epithelialization of RPCs, tubule formation and extension of spatial structure. It is well known that renal tubules need a certain three-dimensional space environment to grow. Single-layer RPCs culture can produce a large number of differentiated tubular epithelial cells. Increasing the number of RPCs layers will form multiple layers of parallel tubular epithelial cells, effectively expanding the spatial environment required for tubular growth. The superstructure formed by the accumulation of RPCs can form an organelle structure under in vitro conditions. This superstructure can be used to study the formation process of organelles under controllable in vitro conditions.

[0006] Chronic kidney disease is a progressive deterioration of renal function, common in elderly kidneys, kidneys with acute immune rejection or vascular rejection, and late stage of kidney transplantation. Its histological characteristics include glomerular sclerosis and tubulointerstitial fibrosis. There is currently no ideal intervention method, and it is a problem that needs to be solved urgently. Studies have found that the origin of interstitial fibroblasts in renal fibrosis is multi-source, including fibroblasts from the primary site, tubular epithelial cells involved in the epithelial-mesenchymal transition process, and bone marrow hematopoietic stem cells. After unilateral ureteral obstruction, the number of RPCs increased significantly, and most of them were PCNA positive. Laminin staining studies found that RPCs were adjacent to the damaged tubular basement membrane and highly expressed vimentin, heat shock protein, α-smooth muscle actin, etc., but the content of BrdU in kidney tissue did not change significantly, indicating that BrdU+ cells in the interstitium are likely to be derived from RPCs through the epithelial-mesenchymal transition pathway. This phenotypic transformation is related to the pathogenesis of renal interstitial fibrosis. The expression of laminin and the absence of basement membrane components indicate that there is damage to the tubular basement membrane in the kidneys of unilateral ureteral obstruction. RPCs are likely to express some proteases, such as MMP-2 and MMP-9, during the degradation of the tubular basement membrane. These proteases can specifically degrade type IV collagen and laminin (the basic components of the tubular basement membrane). Studies have confirmed that in the process of renal fibrosis, RPCs have higher migration, proliferation, and differentiation potential than other cells, making them transformed into fibroblast-like cells.

[0007] At present, there are not enough methods for isolating renal progenitor cells, and conventional isolation methods all cause damage to the kidneys, which are not suitable for promotion and application. In addition, there are not enough studies on the use of renal progenitor cells to treat renal injury, especially the research on drugs with good therapeutic effects, which needs further expansion and research. Summary of the invention

[0008] The present invention provides a method for preparing urine-derived renal progenitor cells. The method is simple to separate and prepare, and the progenitor cells can be used for treatment after proliferation and expansion after separation.

[0009] The method described mainly comprises the following steps:

[0010] Take the morning urine and store it in the preservation solution. Centrifuge the urine sample slowly at 400×g and wash the precipitate twice with phosphate buffered saline (PBS) containing antibiotics and antimycotics. Resuspend the cells in fresh USC medium. Inoculate in a 48-well plate, culture, and gently change the medium. On the 7th day of culture, the cells can form tiny cell colonies. After another week of culture, they can be passaged. Inoculate on a 6-well culture plate coated with gelatin or matrigel. Then inoculate in a 24-well plate and change the medium every 2 days. Passage: Wash the cells once with PBS and gently dissociate them into single cells with 0.25% trypsin / EDTA, and then inoculate them again on the cell culture dish. Cryopreservation: Use 0.25% trypsin / EDTA to detach the cells. Mix the detached cells with freezing medium (FBS 60%, REGM medium 30%, 10% dimethyl sulfoxide (DMSO)) and put them into cryopreservation tubes.

[0011] USC medium consisted of medium A and medium B in a 1:1 ratio. Medium A consisted of high-glucose Dulbecco's modified medium (DMEM; HyClone) + non-essential amino acids (NEAA) solution (Gibco) + Glutamax (Gibco) + 10% fetal bovine serum (FBS; Gibco). Medium B consisted of SingleQuot Kit CC-3191 and CC-4172 renal epithelial cell growth medium (REGM) supplements (Lonza).

[0012] Furthermore, the present invention provides the use of urine-derived renal progenitor cells in the preparation of a drug for treating renal disease caused by ischemia-reperfusion injury.

[0013] The drug can be in oral dosage form.

[0014] Furthermore, the drug also contains a pharmaceutically acceptable carrier.

[0015] Specifically, for oral, buccal and sublingual administration, acceptable solid dosage forms include: powder, suspension, granule, tablet, pill, capsule, soft capsule and capsule. Its preparation method is, for example, mixing one or more compounds of the present invention, or pharmaceutically acceptable salt or its tautomer, adding at least one additive, such as starch or other additives. Suitable additives include: sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymer or glyceride. Alternatively, the oral dosage form may contain other components to assist administration, such as inert diluents, or lubricants such as magnesium stearate, or preservatives such as parabens or sorbic acid, or antioxidants such as ascorbic acid, vitamin E or cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavoring agents or aromatics. Tablets and pills may be further treated with suitable coating materials known in the art.

[0016] Examples of suitable pharmaceutically acceptable wetting agents or surfactants include, but are not limited to, amphoteric, nonionic, cationic or anionic molecules. Suitable surfactants include, but are not limited to, polysorbates, sodium dodecyl sulfate (sodium lauryl sulfate), dodecyl dimethylamine oxide, sodium docusate, cetyl trimethyl ammonium bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol, N, N-dimethyl dodecylamine-N-oxide, cetyl trimethyl ammonium bromide, polyoxyethylene (10) lauryl ether, surfactants (polyoxyethylene fatty alcohol polyoxyethylene ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol), bile salts (e.g., sodium deoxycholate and sodium cholate), polyoxyethylene castor oil, nonylphenol ethoxylate, cyclodextrin, lecithin, methylbenzethonium chloride, carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, and the like.

[0017] Examples of suitable pharmaceutically acceptable preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride and cetylpyridinium chloride, benzyl bromide, benzyl alcohol, disodium EDTA, phenylmercuric nitrate, phenylmercuric acetate, ethylmercuric sodium thiosalicylate, thimerosal, acetate and phenylmercuric borate, polymyxin B sulfate, chlorhexidine, methyl and propyl parabens, phenylethyl alcohol, quaternary ammonium chloride, sodium benzoate, sodium propionate, stabilized chlorine oxygen complex (stabilized oxychloro complex), sorbic acid or mixtures thereof. Preferred pharmaceutically acceptable preservatives include disodium EDTA (disodium ethylenediaminetetraacetic acid) and benzalkonium chloride or mixtures thereof. In one embodiment, the pharmaceutically acceptable preservative is present in an amount of about 0.01% to about 2.0% (weight / volume), preferably about 0.05% to about 1% (weight / volume).

[0018] Examples of suitable pharmaceutically acceptable isotonicity regulators include, but are not limited to, D-mannitol, glucose, glycerol, sodium chloride, potassium chloride, calcium chloride, and magnesium chloride, or any combination thereof. Various nitrates, citrates, acetates, or mixtures thereof may also be used. In one embodiment, the pharmaceutically acceptable isotonicity regulator is present in an amount of about 0.1% to about 1.0% (weight / volume), preferably about 0.5% to about 1% (weight / volume).

[0019] In order to better maintain the activity of renal progenitor cells, the present invention uses the cells as target cells for screening, and obtains the anti-hypoxia peptide HR-8 that specifically inhibits the damage of renal progenitor cells to hypoxia through library screening. The amino acid sequence of the peptide is shown in SEQ ID NO: 1.

[0020] Furthermore, the present invention also provides the use of the anti-hypoxia peptide HR-8 in preparing a culture medium for inhibiting the damage of hypoxic environment to renal progenitor cells.

[0021] Specifically, the culture medium is a culture medium suitable for kidney progenitor cells.

[0022] Further, the culture medium is preferably USC culture medium, which is composed of culture medium A and culture medium B in a ratio of 1:1. Culture medium A is composed of high-glucose Dulbecco's modified medium (DMEM; HyClone) + non-essential amino acids (NEAA) solution (Gibco) + glutamate MAX (Gibco) + 10% fetal bovine serum (FBS; Gibco). Culture medium B is composed of SingleQuot Kit CC-3191 and CC-4172 renal epithelial cell growth medium (REGM) supplement (Lonza).

[0023] Furthermore, the present invention also provides a method for inhibiting the damage of hypoxic environment to renal progenitor cells, which comprises using the anti-hypoxia peptide HR-8.

[0024] Furthermore, the concentration of the polypeptide is 10 μmol / L-200 μmol / L.

[0025] The polypeptide is added to the culture medium.

[0026] Furthermore, the polypeptide can be injected or taken orally to achieve the effect of inhibiting damage.

[0027] One aspect of the present invention provides a method for producing a kidney or kidney cells or tissues, the method comprising the step of injecting progenitor cells to produce a kidney or kidney cells or tissues. In addition, this aspect of the present invention promotes or directs progenitor cells to promote angiogenesis. Beneficial Effects

[0028] The present invention provides a method for preparing urine-derived renal progenitor cells, wherein the prepared renal progenitor cells have stem cell characteristics and can be used to prepare cells for treating renal diseases caused by ischemia-reperfusion injury, and have good therapeutic application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Diagram of isolated renal progenitor cell culture.

[0030] Figure 2 The graph shows the effects of each group on renal progenitor cell apoptosis. DETAILED DESCRIPTION

[0031] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not specifically stated, are all conventional methods, equipment, and materials in the art, and can be purchased from the market.

[0032] Example 1 Isolation and culture of renal progenitor cells from human urine

[0033] The volunteers emptied their urine in the first urine in the morning, drank water or tea after emptying their urine, held their urine to 300 ml, and stored it in preservation solution (transported to the laboratory at 4C°, 200 ml of urine preservation solution + double-antibody culture medium from Dayu MEM culture medium); after entering the laboratory, the urine sample was centrifuged slowly at 400×g for 10 minutes (500×g for 5 minutes), and the precipitate was washed twice with phosphate-buffered saline (PBS) containing antibiotics-antimycotics (Thermo Fisher Scientific); the cells were resuspended in fresh USC culture medium; inoculated in 48-well plates, cultured for 4 days, and the culture medium was gently replaced; on the 7th day of culture, the cells could form tiny cell colonies. After another week of culture, they can be passaged; inoculated on 6-well culture plates coated with gelatin or matrix gel (BD Biosciences); then inoculated on 24-well plates (3 ml of culture medium, 5×10 4 cells / well), change the medium every 2 days; Passaging: wash the cells once with PBS, and gently dissociate them into single cells with 0.25% trypsin / EDTA (Thermo Fisher Scientific), and then seed them on the cell culture dish again. Cryopreservation: detach the cells with 0.25% trypsin / EDTA. 5 The isolated cells were mixed with freezing medium (60% FBS, 30% REGM medium, 10% dimethyl sulfoxide (DMSO)) and placed in cryovials (Corning).

[0034] USC medium consisted of medium A and medium B in a 1:1 ratio. Medium A consisted of high-glucose Dulbecco's modified medium (DMEM; HyClone) + non-essential amino acids (NEAA) solution (Gibco) + Glutamax (Gibco) + 10% fetal bovine serum (FBS; Gibco). Medium B consisted of SingleQuot Kit CC-3191 and CC-4172 renal epithelial cell growth medium (REGM) supplements (Lonza).

[0035] The cell surface markers of the cultured cells were analyzed by flow cytometry and found that USCs expressed common surface markers of MSCs, including CD29, CD73, CD44, CD90 and CD146, but did not express endothelial or hematopoietic cell surface markers, including CD31 and CD45. The low expression of human leukocyte antigen (HLA)-DR indicated that USCs had low immunogenicity. Figure 1 shown.

[0036] Example 2 Effect of anti-hypoxia peptide HR-8 on the activity of renal progenitor cells

[0037] The USC cells isolated in Example 1 were randomly divided into 5 groups: normal control group, hypoxia-reoxygenation (HR) group, HR-8 peptide treatment group, HR+HR-8 group, and positive control group. Normal control group: Normal USC cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO. 2 Hypoxia-reoxygenation (HR) group: cells were cultured in hypoxic conditions (containing 5% CO 2 , 1% 0 2 , and 94% N 2 ) for 24 h and then transferred to a 5% C O2 , 21%O 2 , and 94%N 2 , and reoxygenated for 6h under the conditions of 4h; HR-8 peptide treatment group: cells were treated with HR-8 peptide for 4h at a concentration of 100μmol / L, and no hypoxia-reoxygenation treatment was performed; HR+HR-8 group: HR-8 peptide (at concentrations of 10, 100, and 200 μmol / L, respectively) was added for pretreatment 4h in advance, and then hypoxia-reoxygenation treatment was performed (the operation was the same as the HR group); positive control group: Vc (at a concentration of 100μmol / L) was added for pretreatment 4h in advance, and then hypoxia-reoxygenation treatment was performed (the operation was the same as the HR group).

[0038] Cell damage detection: MTT test was used. The cell suspension density was adjusted to 3×10 4 / ml. The treated cell suspension was inoculated into a 96-well cell culture plate and placed in a 5% CO 2 , incubate in a 37℃ constant temperature incubator; after 24h of culture, discard the original culture medium, add fresh culture medium to the three parallel wells of each group, and record this time as 0h; then test once every 24h; add 20μl of 5mg / ml MTT solution to each well, continue to culture for 4h, discard the liquid in the well, add 200μl of dimethyl sulfoxide (DMSO), shake for 10 min, and measure the absorbance at a wavelength of 580nm. Cell activity (%) = absorbance value of experimental group / absorbance value of control group × 100%.

[0039] Table 1 Test results of cell survival rate in each group

[0040] Group Survival rate (%) Normal control group 100.00±2.47 HR-8 peptide treatment group 135.28±6.88 Hypoxia-reoxygenation (HR) group 52.14±3.58 HR+HR-8 group (10 μmol / L HR-8 peptide) 79.54±4.12 HR+HR-8 group (100 μmol / L HR-8 peptide) 87.69±5.64 HR+HR-8 group (200 μmol / L HR-8 peptide) 96.53±3.18 Positive control group 70.53±2.14

[0041] The results are shown in Table 1. Compared with the control group, the cell survival rate in the HR group was significantly decreased (P<0.05). The HR+HR-8 group significantly increased the activity of HR-treated cells after peptide pretreatment in a dose-dependent manner, and the difference was statistically significant compared with the HR group (P<0.05). Without HR treatment, the cell survival rate was significantly enhanced after treatment with HR-8 peptide alone, indicating that the peptide has a strong effect in promoting the proliferation of renal progenitor cells, and the difference was statistically significant compared with the control group (P<0.05).

[0042] Example 3 Effect of anti-hypoxia peptide HR-8 on apoptosis of renal progenitor cells

[0043] Each grouping was as in Example 2. Cell apoptosis was detected by annexin V / PI double staining. Cells in logarithmic growth phase (1×10 5 ), discard the culture medium, digest the cells moderately with trypsin, blow with the culture medium, centrifuge for 15 minutes, and remove the supernatant. Wash twice with incubation buffer, add 0.5ml phosphate buffer (PBS) and blow evenly. Use a 5ml syringe to suck up the cells, forcefully inject into 5ml of 70% ethanol solution (precooled), and seal with a sealing film. Fix overnight at 4°C, collect the fixed cells, wash twice with incubation buffer, resuspend the cells with 500ul incubation buffer, add 5 μl of fluorescein isothiocyanate-labeled annexin V (annexin V-FITC) and 10μl of 50 mg / ml propidium iodide-PBS solution, and incubate at room temperature in the dark for 15 minutes. Analyze with flow cytometer, the excitation light wavelength is 488nm, and the FITC fluorescence is detected with a passband filter with a wavelength of 515nm, and another filter with a wavelength greater than 560nm is used to detect propidium iodide. The results are as follows Figure 2 shown.

[0044] from Figure 2 It can be seen that compared with the apoptosis rate of the control group (4.90±0.93)%, the apoptosis rate of the HR group increased to (23.24±1.18)%, while in the HR+HR-08 group, as the concentration of the peptide increased, the apoptosis rate of the cells decreased with the increase in the dose, and the lowest was reduced to (5.13±0.47)%, which was statistically significant compared with the HR group (P<0.05). Moreover, the apoptosis rate of each group treated with the peptide was lower than that of the positive control group.

[0045] Example 4 Therapeutic effect of renal progenitor cells on ischemia-reperfusion injury

[0046] BALB / c mice aged 7-8 weeks were given a subcutaneous injection of chloral hydrate. After satisfactory anesthesia, a paraspinal incision was made on the back, and each layer of tissue was cut in turn to expose the renal pedicle. The left renal pedicle was ligated for 30 minutes and then opened to establish an ischemia-reperfusion animal model. Sham-operated animals were only anesthetized and opened to expose the renal pedicle, but no renal pedicle ligation was performed. The experimental animals were divided into the following groups: (1) Control group (10 cases): 3 days before ischemia-reperfusion surgery to 4 days after surgery, subcutaneous injection of normal saline, 0.2 mL / d; (2) Renal progenitor cell group (10 cases): 3 days before ischemia-reperfusion surgery to 4 days after surgery, tail vein injection of 2×10 renal progenitor cells isolated in Example 1 5 (3) Peptide-cultured renal progenitor cell group (10 cases): 2×10 renal progenitor cells were obtained by tail vein injection of renal progenitor cells isolated from Example 1 and cultured under the condition of anti-hypoxia peptide HR-8 at a concentration of 200 μmol / L from 3 days before ischemia-reperfusion surgery to 4 days after surgery. 5 / (kg.d). Four weeks later, the chest was opened after anesthesia, and 20mL PBS was perfused through the left ventricle to wash the blood, and then 5mL of 4% paraformaldehyde was used for perfusion fixation. After perfusion, bilateral kidney specimens were immediately taken and placed in 4% paraformaldehyde perfusion fixative. After sectioning, 10 high-power fields (×400) were randomly selected for vascular density detection. The results are shown in Table 2.

[0047] Table 2 Statistical results of microvessel density in each group

[0048] Group Microvessel density Control group 20.3±2.4 Sham operation group 35.8±3.6# Renal progenitor cell group 32.4±2.9# Peptide-cultured renal progenitor cells 34.9±3.2#

[0049] # indicates significant difference compared with the control group (P<0.05)

[0050] It can be seen from the results in Table 2 that the microvessel density of the kidneys injured by ischemia-reperfusion can be significantly increased after treatment with the renal progenitor cells of the present invention.

[0051] HE staining was used to evaluate the histomorphological changes of the kidneys. On the third day of ischemic injury, there was more swelling and necrosis of tubular cells in the saline control group. In contrast, the tubular cell swelling in the kidneys of the renal progenitor cell group and the renal progenitor cell group cultured with polypeptides was lighter than that of the control group. After 4 weeks of injury, the kidneys of the treatment group were well repaired. This indicates that the polypeptide of the present invention has a good repair effect after treating renal progenitor cells.

[0052] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. Although embodiments of the present invention have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for isolating and culturing human urine-derived stem cells (USC), characterized in that The method comprises the following steps: storing fresh urine in a preservation solution at 4°C and transporting it to a laboratory; after entering the laboratory, centrifuging the urine sample at 400×g for 10 minutes and at 500×g for 5 minutes, and washing the precipitate twice with phosphate buffered saline containing antibiotics and antifungals; resuspending the cells in fresh USC culture medium; inoculating the cells in a 48-well plate, culturing for 4 days, and gently replacing the culture medium; on the 7th day of culture, the cells can form tiny cell colonies; after another week of culture, inoculating the cells in a 6-well culture plate coated with gelatin or matrix gel for culture; and then inoculating the cells in a 24-well plate with 3 ml of culture medium and 5×10 4 cells / well, medium change every 2 days; Passaging: Wash cells once with PBS and gently dissociate into single cells with 0.25% trypsin / EDTA, then re-seed on the cell culture dish; Cryopreservation: Separate cells with 0.25% trypsin / EDTA; Place 5×10 5 The separated cells are mixed with freezing medium: FBS 60%, REGM medium 30%, and 10% dimethyl sulfoxide, and then placed in a cryopreservation tube for freezing to obtain urine-derived stem cells; the urine preservation medium is a MEM medium supplemented with double antibodies; the USC medium is composed of medium A and medium B in a ratio of 1:1, medium A is composed of high-glucose Dulbecco's modified medium + non-essential amino acids (NEAA) solution + glutamate MAX + 10% fetal bovine serum; medium B is composed of SingleQuot Kit CC-3191 and CC-4172 renal epithelial cell growth medium supplement; wherein the urine-derived stem cells are pre-cultured with the anti-hypoxia peptide HR-8, the amino acid sequence of the peptide is shown in SEQ ID NO: 1, and the concentration of the peptide is 10μmol / L-200μmol / L.

2. An anti-hypoxia peptide HR-8 for promoting the proliferation of urine-derived stem cells and reducing the apoptosis rate, characterized in that The amino acid sequence of the peptide is shown in SEQ ID NO:

1.

3. Use of the anti-hypoxia peptide HR-8 as claimed in claim 2 in preparing a culture medium for promoting the proliferation of urine-derived stem cells and reducing the apoptosis rate.

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