Application of urine-derived stem cell culture medium in the preparation of drugs for retinal ischemic diseases

Abstract

The present invention belongs to the field of biomedicine technology and specifically relates to the use of a urine-derived stem cell culture medium in the preparation of a drug for retinal ischemic diseases. The present invention extracts urine-derived stem cells from urine and cultures them in a specific culture medium to obtain a urine-derived stem cell culture medium. The culture medium is then used to construct an OGD / R injury model for R28 cells and treat the cells with a specific concentration of the urine-derived stem cell culture medium. The culture medium has a protective effect on OGD / R-induced R28 cells, and the therapeutic effect is found to be concentration-dependent. Therefore, the urine-derived stem cell-conditioned culture medium can be further developed into a drug for preventing or delaying the progression of eye diseases, particularly for treating retinal diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of a urine-derived stem cell culture medium in the preparation of a drug for retinal ischemic diseases. Background Art

[0002] Urine-derived stem cells (USCs) are a type of cell with a high degree of self-replication and multidirectional differentiation capacity that can be obtained from fresh urine of animals such as humans, pigs, monkeys, and rabbits through centrifugation. Under certain conditions, USCs can differentiate into multiple cell types, including bone cells, adipocytes, chondrocytes, and neurons. USCs are widely available and easy to obtain. Unlike adipose-derived stem cells (ADSCs) and bone marrow mesenchymal stem cells (BMSCs), which require invasive, enzymatic methods, USCs can be isolated from urine using non-invasive, non-enzymatic methods, using an economical and convenient method. An average of 0.7-1.4 USCs can be isolated and extracted from every 100 mL of urine. In terms of proliferation and differentiation potential, USCs have a stronger proliferation capacity and better adipogenic ability than BMSCs. Compared to ADSCs, USCs have a higher proliferation rate, stronger colony-forming ability, a higher expression rate of stem cell markers, a higher efficiency in suppressing immune cell activation, and exhibit higher rates of myogenic, neurogenic, and endogenous differentiation. Since their first successful isolation from Zhang Yuanyuan's laboratory, USCs have demonstrated potential for treating diseases such as kidney disease, digestive disease, diabetes, and its complications.

[0003] As windows to the soul, the eyes are crucial to our daily lives. While our eyes allow us to perceive the world in all its splendor, eye-related diseases have been on the rise in recent years, becoming a major factor affecting vision. Among these, glaucoma, age-related macular degeneration, diabetic retinopathy, and central retinal artery or vein occlusion are the most prominent. Retinal ischemia is a major cause of visual field loss and decreased vision, and a potential mechanism underlying these diseases. The pathophysiological process of retinal ischemia is initially characterized by insufficient blood supply due to high intraocular pressure. Reperfusion restores blood flow and reoxygenation, but this is often accompanied by severe inflammation, stress, and tissue damage. Following disease onset, the retina's regenerative capacity is limited, and existing treatments are limited, primarily relying on intraocular injections, eye drops, or surgery to halt disease progression, with limited efficacy. Therefore, a novel treatment approach is urgently needed. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention extracts urine-derived stem cells from urine, and subcultures them through a specific culture medium to obtain a urine-derived stem cell culture medium. Then, by constructing an OGD / R injury model of R28 cells (retinal progenitor cells), the pathophysiological process of retinal ischemia-reperfusion is simulated in vitro, and a specific concentration of urine-derived stem cell culture medium is used for treatment. The culture medium has a protective effect on OGD / R-induced R28 cells, and the therapeutic effect is found to be concentration-dependent. The method is used to prepare a drug for protecting eye cells, which can be further developed into a drug for preventing glaucoma or delaying the deterioration of glaucoma.

[0005] Based on the above research, an embodiment of the present invention provides the use of a urine-derived stem cell culture medium in the preparation of a drug for retinal ischemic diseases.

[0006] Based on the same inventive concept, an embodiment of the present invention further provides a pharmaceutical composition for treating retinal ischemic diseases, wherein the pharmaceutical composition comprises a urine-derived stem cell culture medium.

[0007] Furthermore, the preparation comprises at least one pharmaceutically acceptable excipient;

[0008] The auxiliary material is one or more of a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative and a fragrance.

[0009] Based on the same inventive concept, an embodiment of the present invention further provides a method for preparing a urine-derived stem cell culture medium, the preparation method specifically comprising the following steps:

[0010] S1: Midstream urine was collected and centrifuged, resuspended, and washed multiple times to obtain cell microparticles;

[0011] S2: culturing the cell microparticles in a primary culture medium and a proliferation culture medium successively to obtain urine-derived stem cells;

[0012] S3: After removing the culture medium of the urine-derived stem cells, rinsing with PBS, adding proliferation medium and exosome-free fetal bovine serum for culturing, collecting the culture medium and concentrating it to obtain urine-derived stem cell culture medium.

[0013] Furthermore, the primary culture medium is prepared by adding 10% fetal bovine serum, 1% antibiotic-antimycotic agent and DMEM / F-12 to the REGM SingleQuot kit and mixing them evenly;

[0014] The proliferation medium is prepared by adding 10% FBS, 1% Antibiotic-Antimycotic, 1% GlutaMAX, 1% NEAA, 5 ng / mL bFGF, 5 ng / mL PDGF-BB, and 5 ng / mL EGF to DMEM / F12 and mixing with the primary medium in a ratio of 1:1;

[0015] Furthermore, the culture conditions are: 37°C, 5% CO2.

[0016] Furthermore, the specific process of culturing the cell microparticles in the primary culture medium and the proliferation culture medium is as follows:

[0017] The cell microparticles were suspended in primary culture medium, transferred to well plates and cultured for 48 h, followed by addition of primary culture medium and re-plating;

[0018] After plating, the cells were cultured for 96 hours, and part of the primary culture medium was removed. An equal amount of proliferation culture medium was added, and the cells were cultured for another day and replaced with complete proliferation culture medium. When the cell density reached 80-90%, the proliferation culture medium was used for subculture to obtain urine-derived stem cells in the logarithmic phase.

[0019] Furthermore, the preparation method of the exosome-free fetal bovine serum is:

[0020] Fetal bovine serum was centrifuged twice at 10,000 g for 70 min each time and then filtered through a 0.22 μm sterilizing filter.

[0021] Beneficial effects:

[0022] The present invention proposes for the first time the application of urine-derived stem cell culture medium in drugs related to retinal ischemic diseases, which can reduce the damage to retinal progenitor cells caused by oxygen-glucose deprivation and recovery, so that the prepared drug has a protective effect on retinal progenitor cells; the urine-derived stem cells can secrete related growth factors and extracellular vesicles through paracrine effects. The vesicles contain a large number of important substances such as membrane proteins and miRNAs from urine-derived stem cells, which can regulate the cell cycle, promote the proliferation of damaged cells, and repair damaged cells by inhibiting oxidative stress and inflammation. Therefore, the urine-derived stem cell conditioned medium can be further developed into a drug to prevent or delay the deterioration of eye diseases, and is particularly useful for treating retinal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Figures showing correlation between the detection of R28 cells at different recovery time points after oxygen-glucose deprivation provided in an embodiment of the present invention; A) is a graph showing the positive staining analysis of PI (red) / DAPI (blue) in R28 cells, scale bar = 50 μm; B) is a graph showing the statistical analysis of PI-positive cells, and C) is a graph showing the statistical analysis of CCK-8 viability detection in R28 cells;

[0025] Figure 2 Figures showing the effects of different concentrations of USC-CM on OGD / R R28 cell damage in an embodiment of the present invention; A) is a graph showing PI (red) / Hoechst (blue) positive staining analysis of R28 cells; B is a statistical analysis of PI-positive cells; and C is a statistical analysis of CCK-8 viability assay of R28 cells.

[0026] Figure 3 Western Blot analysis results provided in Example 3 of the present invention; A) is a WB band diagram of protein expression in each group; B) is a semi-quantitative analysis result diagram of p-Akt / Akt results in each group; C) is a semi-quantitative analysis result diagram of Bcl-2 / Bax results in each group; D) is a semi-quantitative analysis result diagram of cl-caspase-3 / GAPDH results in each group;

[0027] Figure 4 Flow cytometry analysis results provided in Example 3 of the present invention; A) is a graph showing the cell apoptosis results of each group; B) is a statistical analysis result of the apoptosis rate of R28 cells;

[0028] Figure 5 The expression of PTEN, an upstream molecule of the Akt pathway, in each group provided in Example 3 of the present invention. A) is a Western blot image of PTEN and GAPDH; B) is a semi-quantitative analysis of PTEN and GAPDH. DETAILED DESCRIPTION

[0029] In order to more clearly illustrate the technical content of the present invention, it is described in detail with reference to specific embodiments and drawings. Obviously, the enumerated embodiments are only preferred implementation plans of the present technical solution. Other technical solutions that can be obviously derived by those skilled in the art based on the disclosed technical content still fall within the scope of protection of the present invention.

[0030] The definitions of specific terms are described in more detail below. This disclosure is not intended to be limited in any way by the exemplary list of substitutes described herein. In the embodiments of the present invention, the chemical reagents used can be purchased or prepared by existing preparation methods, and the instruments and equipment used are conventional equipment in the prior art.

[0031] Example 1

[0032] Preparation of urine-derived stem cell culture medium:

[0033] Urine samples were collected from five healthy male volunteers aged between 24 and 35 years. After signing the informed consent form, they were generally informed of the collection process one day in advance. The urine collection container was sterilized in advance with high temperature and high pressure, and double antibodies were added in advance to ensure that the cells were not contaminated in the future. The midstream urine was taken and placed in a biosafety cabinet and quickly divided into sterile 50mL centrifuge tubes with corresponding labels. Start the centrifuge, centrifuge at 400g centrifugal force for 10 minutes, carefully remove the supernatant, and leave 1mL of microparticle sediment at the bottom of the tube. Gently resuspend these microparticles in the remaining 1mL of urine, and add 10mL of sterile PBS to the centrifuge tube. Start the centrifuge again, centrifuge at 200g centrifugal force for 10 minutes, discard the supernatant, and only 0.2mL of cell microparticles remain. Repeat the washing of the microparticles in the urine twice.

[0034] Preparation of primary culture medium: 10% fetal bovine serum (FBS; Gibco), 1% antibiotic-antimycotic (Gibco), and DMEM / F-12 were added to the REGM SingleQuot kit (Lonza, USA) and mixed thoroughly. Preparation of proliferation culture medium: 10% FBS, 1% antibiotic-antimycotic, 1% GlutaMAX, 1% NEAA, 5 ng / mL bFGF, 5 ng / mL PDGF-BB, and 5 ng / mL EGF were added to DMEM / F12 and mixed 1:1 with the primary culture medium.

[0035] At room temperature, resuspend the obtained cell microparticles in 3 mL of primary culture medium, transfer them evenly to 12-well plates, and culture them at 37°C in a humidified atmosphere with 5% CO₂ for 48 hours. Without removing any culture medium, add 1 mL of primary culture medium again. Continue culturing for approximately 96 hours. Remove 1 mL of primary culture medium and add 1 mL of proliferation medium. Replace the entire culture medium with complete proliferation medium every other day. Carefully clean the surface of the wells. Because USC colonies are initially small and difficult to detect, carefully observe and count them under a microscope. Passage the cells when the cell density reaches 80-90%. Continue culturing the passaged USCs in proliferation medium with daily medium changes. Keep track of cell status and gradually expand the culture. The USCs in the 12-well plates are digested and passaged into T25 medium, typically taking no more than 2 minutes. After digestion, the cells are centrifuged, the wastewater removed, and resuspended for passage.

[0036] Fetal bovine serum (FBS) was centrifuged twice at 10,000g for 70 minutes each time and filtered through a 0.22μm sterile filter to obtain exosome-free FBS. USCs growing in the logarithmic phase of culture were selected and cultured to a density of approximately 80%. The remaining culture medium was removed and the tubes were gently rinsed twice with 5mL of PBS along the sides. Then, 12mL of proliferation medium supplemented with 10% exosome-free FBS was added to the T75 tubes. The tubes were incubated at 37°C for 24 hours. The culture medium was collected and filtered, concentrated 10-fold using a 50mL centrifugal filter at 15,000g for 30 minutes. The approximately 1.5mL of fluid remaining at the top of the ultrafiltration tube was used as urine-derived stem cell-conditioned medium (USC-CM). If not in use, it can be stored at -80°C. Sterility was ensured throughout the entire process, and strict aseptic procedures were followed.

[0037] Example 2

[0038] Effects of USC-CM on OGD / R-induced R28 cell injury

[0039] Prepare R28 culture medium: mix DMEM low-glucose culture medium, fetal bovine serum (FBS), and penicillin / streptomycin solution in the ratio of 89%, 10%, and 1% in sequence.

[0040] Select cells that are in good condition after 3-8 generations of recovery for modeling. When the cell density reaches between 80-90%, transfer them to a clean bench. Remove the culture medium and rinse the culture dish with PBS. Add PBS along the sides of the dish to prevent cells from falling off during rinsing. After rinsing twice, replace the culture medium with sugar-free culture medium. Wipe the modeling device with alcohol in advance and sterilize it under UV light for half an hour. When assembling the modeling device, place a 3.5mm diameter culture dish at the bottom and add an appropriate amount of sterilized deionized water. Place the culture dishes of all experimental groups on the upper rack, ensuring that the rack is level. Gently close the lid, observing the position of the snaps as you rotate the lid to ensure the device is airtight. After assembly, open the nitrogen tank, adjust the setting, and begin injecting nitrogen at a rate of 5mL / min, timing for 5 minutes. After inflation is complete, first move the catheter on one side to the other opening to completely seal the modeling device. Then close the nitrogen tank. The inflated modeling device was transferred to a constant temperature incubator. After approximately 2.5 hours, the modeling was complete. The modeling device was opened and transferred to a clean bench. The sugar-free culture medium was replaced with normal culture medium. The cells were continuously observed under a microscope and further processed according to the subsequent experimental design.

[0041] The cells were treated according to the modeling steps described above. The cells were divided into a control group, an OGD / R group, and an OGD / R+CM group. After 4 hours of reoxygenation, all culture media were replaced, and the amount of PI staining required for all experimental groups was calculated. The fluorescent dye PI (propidium iodide) is a nuclear staining reagent that stains DNA and is commonly used to detect cell apoptosis. It is an analog of ethidium bromide and emits red fluorescence after intercalating into double-stranded DNA. Although PI cannot pass through the membranes of living cells, it can penetrate damaged cell membranes and stain nuclei. It is often used in conjunction with Hoechst live cell staining solution to stain both live and dead cells. The PI staining analysis steps include: Cells seeded in 6-well plates were divided into a control group and other intervention groups according to the experimental design. The cells were then modeled and treated with stem cell-conditioned medium according to the steps described above. 1 mL of PI staining solution was added to each well. The culture plate surface was covered with aluminum foil and allowed to stand for approximately 15 minutes. After staining is completed, wash off the staining solution, add 500ul of 4% paraformaldehyde, fix for 15 minutes (avoid light), pour out the fixative, add 1mL PBS to the culture plate and place it on a shaker for 3 minutes. Repeat this process twice, and pay attention to avoid light. Add 1mL Hoechst staining solution (concentration of 1:1000) to each well again and stain for 5 minutes in the dark. Repeat washing with PBS twice, add PBS again to keep the cells moist. Then add glycerol to seal the slides. Store in a box completely away from light and transfer to a fluorescence microscope room for photography. For detailed results, see Figure 1 A and Figure 2 A.

[0042] CCK-8 solution (Cell Counting Kit-8, abbreviated as CCK-8 kit, is a rapid, highly sensitive, non-radioactive colorimetric detection kit based on WST-8 and widely used in cell proliferation and cytotoxicity. CCK-8 solution can be added directly to the cell sample without the need to pre-mix various components. WST-8 can be reduced by some dehydrogenases in the mitochondria to produce orange-yellow formazan in the presence of electron coupling reagents). Mix the CCK-8 solution into the complete culture to ensure a final concentration of 10%. Then use a multi-channel pipette to add 100 μl of mixed culture medium to each well, and let CCK-8 react with cells in the incubator for 2 to 4 hours. After incubation, turn on the microplate reader, adjust the program, select the target area, and finally save the light absorbance value at 450 nm. Calculate the survival rate of cells in all experimental groups according to the formula.

[0043] Calculation formula

[0044] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0045] Inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%

[0046] As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution);

[0047] Ac: absorbance of control well (containing cells, culture medium, and CCK-8 solution, but no drug);

[0048] Ab: absorbance of blank wells (containing culture medium and CCK-8 solution, but not cells or drugs).

[0049] All results are shown as mean ± SD, and the means were analyzed by one-way ANOVA (One-way ANOVA) using GraphPad Prism 7. The differences were considered statistically significant when p < 0.05.

[0050] According to the above experimental steps, an in vitro OGD / R model was successfully constructed. The results showed that OGD / R treatment significantly reduced cell viability. In order to determine the optimal time point for modeling, different recovery times were performed in sequence, and it was found that the degree of damage was time-dependent. CCK-8 cell viability assay was used to detect R28 cells at different recovery time points (0, 2, 4, 8, and 12 hours) after oxygen glucose deprivation. The results are as follows: Figure 1 As shown in Figure 2, compared with the Control group, the degree of injury was the greatest at 4 h of recovery (P < 0.001 Figure 1 (B, C) At 2 hours of recovery, the degree of injury was still above 40%. At 0 hours of recovery, no statistical difference was observed compared to the normal group. At 12 hours, the degree of injury was reduced compared to the previous time points. These results indicate that the in vitro OGD / R model was successfully established, and 4 hours was selected as the recovery time point for subsequent experiments.

[0051] The obtained USC-CM was mixed with 10% fetal bovine serum and set to contain different concentration gradients, specifically 0, 1:100, 1:50, 1:20, 1:10, 1:5 and 1:1. The R28 cells after OGD / R were treated with the above-mentioned different concentrations of USC-CM. The results of PI staining, statistical analysis of PI-positive cells and CCK-8 showed that USC-CM had a protective effect on OGD / R-induced R28 cells and the therapeutic effect was found to be concentration-dependent; there was no significant difference at the two concentrations of 1:50 and 1:20, but the protective effect disappeared when the dose was increased to 1:1. At doses of 1:5 and 1:10, USC-CM significantly repaired damaged cells (P<0.001). The specific results are as follows: Figure 2 As shown in B and C.

[0052] Example 3

[0053] After cells reached approximately 70%-80% of their growth, they were treated with OGD for 2.5 hours and recovered to the corresponding time point. The modeling group (OGD / R) and the normal control group (Control) were removed from the incubator, the culture medium in the culture dishes was aspirated, and the cells were washed twice with ice-cold phosphate buffered saline. Subsequently, USC-CM (OGD / R+CM) was added to each small culture dish at a concentration of 1:10. Another group was treated with LY294002 for 1 hour before modeling (OGD / R+CM+LY294002). LY294002 was initially powdered and dissolved in DMSO to a final concentration of 50 μM / L. The expression of related proteins and apoptosis were analyzed using Western blot and an early apoptosis detection kit (Annexin V-FITC / PI, Cell Apoptosis Kit, Dalian Meilun). The antibodies used in the Western blot analysis process are Akt (Wanlei Bio), p-Akt (Wanlei Bio), Bcl-2 (Wanlei Bio), Caspase-3 (Abcam), PTEN (Cell Signaling Technology), and GAPDH (Wanlei Bio). The Western Blot result bands were analyzed using the Image J image software system to measure the average absorbance value, the average absorbance value of the target molecule was compared with the average absorbance value of GAPDH, and the positive cells in the morphological pictures were counted and counted using the Image J image software analysis system. All results are shown as Mean ± SD, and the mean was subjected to one-way ANOVA (One-way ANOVA) using GraphPad Prism 7, with p < 0.05 indicating that the difference is statistically significant. For detailed results, see Figure 3-Figure 5 .

[0054] like Figure 3 As shown in the figure, OGD treatment significantly reduced the expression of p-Akt, while the expression of apoptosis-related factor cl-Caspase-3 was significantly increased. Bcl-2 / bax decreased, while p-Akt was significantly increased and the expression of apoptosis-related factor cl-Caspase-3 was significantly decreased in the OGD / R+CM group. Bcl-2 / bax expression increased, while the protective effect of USC-CM was inhibited in the group with the addition of Akt blocker LY294002. Figure 4As shown in the results, compared with the control, the apoptosis rate of R28 cells treated with OGD / R increased (17.16±1.17% vs 5.84±2.95%; p<0.001). After the addition of USC-CM, the apoptosis rate decreased from 17.16% to 11.55% (p<0.05). More importantly, the Akt blocker LY294002 inhibited the protection of R28 cells by USC-CM. Based on the above results, the expression of PTEN, an upstream molecule of the Akt pathway, in each group was detected. The results showed that the expression of PTEN was significantly increased in the OGD / R group, and the expression of PTEN was decreased after USC-CM (as shown in the figure). Figure 5 , p<0.05).

[0055] It can be seen from this that urine-derived stem cell conditioned medium can reduce the damage to retinal progenitor cells caused by oxygen-glucose deprivation and recovery. USC-CM reduces OGD / R-induced apoptosis of R28 cells by regulating the PTEN / Akt pathway. It can be used to prepare drugs to protect eye cells and can be further developed into drugs to prevent glaucoma or delay the progression of glaucoma in the future.

[0056] The embodiments described above are only preferred specific implementation methods of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solutions and concepts of the present invention within the technical scope of the present invention, and they should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing a urine-derived stem cell culture medium for preventing and treating retinal ischemic diseases, characterized in that: The preparation method specifically comprises the following steps: S1: Midstream urine was collected and centrifuged, resuspended, and washed multiple times to obtain cell microparticles; S2: The cell microparticles are suspended in a primary culture medium, transferred to a well plate and cultured for 48 hours, after which primary culture medium is added and the cells are plated again; after plating for 96 hours, part of the primary culture medium is removed, and an equal amount of proliferation culture medium is added. The cells are cultured for another day and then replaced with complete proliferation culture medium. When the cell density reaches 80-90%, the proliferation culture medium is used for subculture to obtain urine-derived stem cells in the logarithmic phase; wherein the primary culture medium is prepared by adding 10% fetal bovine serum, 1% antibiotic-antimycotic agent, and DMEM / F-12 to the REGM SingleQuot kit and mixing evenly; the proliferation culture medium is prepared by adding 10% FBS, 1% Antibiotic-Antimycotic, 1% GlutaMAX, 1% NEAA, 5 ng / mL bFGF, 5 ng / mL PDGF-BB, and 5 ng / mL EGF to DMEM / F12 and mixing with the primary culture medium in a 1:1 ratio; S3: After removing the culture medium of the urine-derived stem cells, the cells were rinsed with PBS, and proliferation medium and exosome-free fetal bovine serum were added for culturing at 37° C. and 5% CO 2 for 24 h. The culture medium was collected and concentrated to obtain a urine-derived stem cell culture medium; The preparation method of the exosome-free fetal bovine serum is as follows: Fetal bovine serum was centrifuged twice at 10,000 g for 70 min each time and then filtered through a 0.22 μm sterilizing filter.

2. Use of the urine-derived stem cell culture medium prepared by the preparation method according to claim 1 in the preparation of a drug for treating retinal ischemic diseases.

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