Application of homing and drug effect enhanced mesenchymal stem cells

Abstract

The invention relates to application of homing and drug effect enhanced mesenchymal stem cells, and belongs to the technical field of mesenchymal stem cells. According to the invention, the human umbilical cord mesenchymal stem cells are selected, and the human umbilical cord mesenchymal stem cells are pretreated by adding resveratrol in an in-vitro culture stage, so that the in-vivo kidney homing rate and immune microenvironment regulation capability of the human umbilical cord mesenchymal stem cells are improved. Wherein the resveratrol is a natural polyphenol compound, and the chemical name of the resveratrol is 3, 5, 4 '-trihydroxy stilbene. The mesenchymal stem cells pretreated by the method show significantly improved kidney homing rate in a lupus nephritis mouse model, and the ability of inhibiting kidney CD4 + T cell infiltration is significantly enhanced. The clinical application effectiveness of the mesenchymal stem cells is improved, and a new technical strategy is provided for treating lupus nephritis and other types of nephritis by using the mesenchymal stem cells.

Description

Technical Field

[0001] This invention relates to the field of mesenchymal stem cell technology, and more particularly to the application of a homing and drug-enhancing type of mesenchymal stem cell. Background Technology

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease, and lupus nephritis is one of its serious complications and a major risk factor for morbidity and death. Currently, the primary goal of lupus nephritis treatment is to prevent chronic kidney disease and end-stage renal disease. Clinically, anti-inflammatory drugs are often used to rapidly relieve inflammation in the kidneys, combined with highly effective immunosuppressants to block the autoimmune response. However, long-term treatment often has poor efficacy and frequently results in serious drug side effects or toxicity. Therefore, there is an urgent need for more effective new therapies for lupus nephritis.

[0003] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cell located in the mesoderm, primarily found in connective tissue and organ stroma. Under suitable conditions, they can differentiate into various cell types, including skeletal muscle, cardiac muscle, fat, cartilage, and bone. Studies have shown that MSCs possess biological properties such as anti-fibrosis, anti-inflammation, anti-apoptosis, immunomodulation, and pro-angiogenesis, thus finding applications in the treatment of some critical or refractory diseases, such as graft-versus-host disease (GvHD), acute respiratory distress syndrome (ARDS), and systemic lupus erythematosus (SLE).

[0004] MSCs possess homing characteristics. However, the expression levels of chemokine receptors associated with homing on MSCs are typically low. Furthermore, the homing efficiency of MSCs after systemic administration is not high due to factors such as pulmonary capillary network retention and complex in vivo pathological environments, directly limiting their clinical application and translation. Studies have shown that after intravenous injection, the homing efficiency of MSCs in the bone, bone marrow, and skin of children with osteogenesis imperfecta is less than 1%. In addition, the immunomodulatory effects of MSCs require induction by inflammatory cytokines. However, the inflammatory state in the disease microenvironment is dynamic, and at low inflammation levels, the immune response of MSCs cannot be activated. This means that MSCs may exert immunosuppressive effects when pro-inflammatory cytokine concentrations are high, or exacerbate inflammatory responses when pro-inflammatory cytokine concentrations are low. This bidirectional regulatory characteristic poses a challenge to the controllability of therapeutic effects.

[0005] Compared to traditional methods such as genetic engineering or small molecule compound processing, traditional Chinese medicine (TCM), a treasure of TCM, has shown enormous application potential in its active extracts—TCM monomers. Resveratrol (Res) is a non-flavonoid polyphenolic phytoalexin with a stilbene structure. It was isolated in 1940 by Japanese researchers from *Veratrum album*, a plant in the Liliaceae family, and subsequently discovered in the roots of *Polygonum cuspidatum*, a plant in the Polygonaceae family. As a natural drug, resveratrol, due to its antioxidant, anti-inflammatory, and anticancer properties, can protect various tissues from serious damage caused by acute or chronic injury. Studies have shown that resveratrol can enhance the survival, self-renewal, and delay aging of mesenchymal stem cells (MSCs), thus resveratrol has the potential to enhance the in vivo homing efficiency of MSCs and improve their clinical application and transformation. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a homing and pharmacologically enhanced application of mesenchymal stem cells (MSCs). This technology can significantly improve the homing rate of MSCs in the kidneys and their ability to regulate the immune microenvironment in vivo, thereby enhancing the effectiveness of MSCs in clinical applications.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] First, the present invention provides a traditional Chinese medicine monomer for improving the in vivo renal homing rate and immune microenvironment regulation ability of human mesenchymal stem cells, wherein the monomer is resveratrol.

[0009] In this invention, resveratrol is a natural polyphenol compound with the chemical name 3,5,4'-trihydroxystilbene. It is mainly found in plants such as grapes, peanuts, and Japanese knotweed, and has a wide range of biological activities.

[0010] Second, the present invention provides a pretreatment culture medium for improving the in vivo renal homing rate and immune microenvironment regulation ability of human mesenchymal stem cells.

[0011] Preferably, the pretreatment culture medium contains the aforementioned traditional Chinese medicine monomer resveratrol.

[0012] Preferably, the concentration of resveratrol in the pretreatment culture medium ranges from 0.1 to 20 μM, and the preferred concentration is 2.5 μM.

[0013] Preferably, the pretreatment medium is based on DMEM / F12 medium.

[0014] Third, the present invention also provides a method for preparing human mesenchymal stem cells that improve the homing rate of the kidneys and the ability to regulate the immune microenvironment in vivo, comprising the following steps: seeding human mesenchymal stem cells into DMEM / F12 medium containing resveratrol and culturing for 24-72 h.

[0015] Preferably, the seeding density of the human mesenchymal stem cells is 10-1. 5 ~10 7 cells / ml.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention provides a traditional Chinese medicine monomer, resveratrol, as a culture medium additive. Pretreatment of human mesenchymal stem cells at a specific concentration (2.5 μM) and time (24 h) successfully improved the kidney homing rate of human mesenchymal stem cells in mice with lupus nephritis and enhanced the inhibitory effect of human mesenchymal stem cells on T cell infiltration in the kidneys of mice with lupus nephritis. This opens up a new avenue for the treatment of lupus nephritis and has important clinical application and translational value in the field of autoimmune disease treatment. Attached Figure Description

[0018] Figure 1 The effect of different concentrations of resveratrol on the cell viability of human umbilical cord mesenchymal stem cells;

[0019] Figure 2 The effects of different concentrations of resveratrol and different pretreatment times on the total mRNA expression levels of CXCR3, IDO, and IL4I1 in human umbilical cord mesenchymal stem cells were investigated.

[0020] Figure 3 The effect of 2.5 μM resveratrol pretreatment for 24 h on the expression levels of total proteins CXCR3, IDO, and IL4I1 in human umbilical cord mesenchymal stem cells;

[0021] Figure 4 Effect of 2.5 μM resveratrol pretreatment for 24 h on the expression level of chemokine receptor CXCR3 on the membrane of human umbilical cord mesenchymal stem cells;

[0022] Figure 5 The effect of 2.5 μM resveratrol pretreatment for 24 h on the migration of human umbilical cord mesenchymal stem cells in response to chemokine CXCL10;

[0023] Figure 6 The effect of 2.5 μM resveratrol pretreatment for 24 h on the regulation of inflammatory T cells in human umbilical cord mesenchymal stem cells;

[0024] Figure 7Effect of 2.5 μM resveratrol pretreatment for 24 h on the kidney homing rate of human umbilical cord mesenchymal stem cells in mice with lupus nephritis;

[0025] Figure 8 Effects of 2.5 μM resveratrol pretreatment for 24 h on the renal immune microenvironment of human umbilical cord mesenchymal stem cells in mice with lupus nephritis. Detailed Implementation

[0026] To provide a further understanding of the purpose, structure, features, and functions of the present invention, detailed descriptions are provided below with reference to specific embodiments.

[0027] Example 1: Effect of resveratrol on MSC cell viability.

[0028] Experimental materials: human umbilical cord mesenchymal stem cells, phosphate-buffered saline (PBS), trypsin solution, DMEM / F12 complete culture medium (90% DMEM / F12 basal medium + 10% serum), DMEM / F12 basal medium, and resveratrol.

[0029] Experimental methods:

[0030] Human umbilical cord mesenchymal stem cell in vitro culture and seeding: Human umbilical cord mesenchymal stem cells were cultured in a culture dish. When the cell confluence was observed to be 80%-90% under an inverted microscope, the original culture medium was removed, the cells were washed once with PBS buffer, and then trypsin solution was added for digestion for 2 min.

[0031] Under an inverted microscope, most human umbilical cord mesenchymal stem cells were observed to float as round dots. An equal volume of DMEM / F12 complete culture medium was added to terminate trypsin digestion. The mixture was collected into a centrifuge tube and centrifuged at 500g for 5 minutes. The supernatant was discarded, yielding a purified human umbilical cord mesenchymal stem cell pellet. The cells were then resuspended in DMEM / F12 complete culture medium.

[0032] After resuspending and counting the cells, use 1×10⁻⁶ cells per well. 4 200 μL of cells were seeded into 96-well plates. After 12 hours of cell adhesion, the original culture medium was removed, and the cells were divided into three groups and given fresh culture medium.

[0033] The control group was given 200 μL of DMEM / F12 basal medium;

[0034] The experimental groups were given 200 μL of DMEM / F12 basal medium with resveratrol concentrations of 0.1, 1, 2.5, 5, 10, 20, 50, and 100 μM.

[0035] The blank control was set as a basal culture medium without cells.

[0036] Each group was set up with 6 replicates and cultured in a cell culture incubator at 37°C and 5% CO2.

[0037] After culturing for 24 hours, the supernatant was discarded, and the cells were washed twice with PBS buffer. 100 μL of LDM / F12 basal medium was added to each well, followed by 10 μL of CCK8 solution. The cells were incubated at 37°C for 2 hours, and the absorbance at 450 nm was measured to calculate cell viability.

[0038] Cell viability (%) = [(OD) 实验孔 -OD 空白孔 ) / (OD 对照孔 -OD 空白孔 )]×100%.

[0039] The results obtained in Example 1 are as follows Figure 1 As shown. Figure 1 This shows the viability of human umbilical cord mesenchymal stem cells after culturing in DMEM / F12 basal medium supplemented with 0.1, 1, 2.5, 5, 10, 20, 50, and 100 μM resveratrol.

[0040] As shown in the figure, the cell viability was around 100% when resveratrol concentrations below 100 μM were added, with no cases of excessively low viability. This indicates that concentrations below 100 μM have no significant effect on the viability of human umbilical cord mesenchymal stem cells.

[0041] Example 2: Exploration of the effects of resveratrol pretreatment on the expression of CXCR3, IL4I1, and IDO genes in MSCs by concentration and time.

[0042] Experimental materials: human umbilical cord mesenchymal stem cells, DMEM / F12 basal culture medium, resveratrol, and mRNA rapid extraction kit.

[0043] Experimental methods:

[0044] After counting the resuspended cells from Example 1, the cells were divided into 8 × 10⁸ cells per well. 4 700 μL of cells were seeded into 24-well plates. After 12 hours of cell attachment, the original culture medium was removed, and the cells were divided into groups and given fresh culture medium.

[0045] The control group was given 700 μL of DMEM / F12 basal medium;

[0046] The experimental groups were given 700 μL of DMEM / F12 basal medium containing 0.25, 1, 2.5, 10, and 25 μM resveratrol.

[0047] Each group was set up with 5 replicates and cultured in a cell culture incubator at 37°C and 5% CO2 for 12, 24, 48, and 72 hours.

[0048] Total mRNA extraction from human umbilical cord mesenchymal stem cells: Cells were lysed, centrifuged, washed, centrifuged again, washed again, centrifuged empty, eluted mRNA, and purified by centrifugation to obtain total mRNA.

[0049] Total mRNA from human umbilical cord mesenchymal stem cells was reverse transcribed into cDNA: The obtained total mRNA could be immediately reverse transcribed into cDNA. The reverse transcription system of the reverse transcription kit was added according to the system shown in Table 1. Reverse transcription was performed using a qPCR instrument under the conditions of 37℃ for 15 min and 85℃ for 5 s to obtain cDNA.

[0050] Table 1 Reverse Transcription System

[0051]

[0052] Real-time quantitative polymerase chain reaction (Real-Time-qPCR) was used to detect human umbilical cord mesenchymal stem cell cDNA using a 96-well fluorescent quantitative plate. Primer sequences are shown in Table 2, reaction systems in Table 3, and qPCR programs in Table 4. The internal control GAPDH gene and the target gene for each sample were amplified under the same conditions. Melting curve analysis was performed after the qPCR reaction to exclude contamination from non-specific qPCR products.

[0053] Use 2 -ΔΔCT The analysis method is as follows: First, subtract the Ct value of the internal reference GAPDH gene from the Ct value of the target gene to obtain ΔCt, that is, ΔCt = Ct. CXCR3 –Ct GAPDH Then, the ΔCt value of the target gene in the experimental group is subtracted from the ΔCt value of the target gene in the control group to obtain ΔΔCt, i.e., ΔΔCt = ΔCt. 实验组 –ΔCt 对照组 The expression level of the target gene in the experimental group was 2 compared to the control group. -ΔΔCt .

[0054] Table 2 Primer sequences for real-time quantitative qPCR

[0055]

[0056] Table 3 Real-time quantitative qPCR reaction system

[0057]

[0058] Table 4 Real-time quantitative qPCR program

[0059]

[0060]

[0061] The results obtained in Example 2 are as follows Figure 2 As shown.

[0062] Figure 2 This study reflects the effects of different concentrations of resveratrol and different pretreatment times on the total mRNA expression levels of CXCR3, IDO, and IL4I1 in human umbilical cord mesenchymal stem cells.

[0063] Using resveratrol concentration 0 and pretreatment time 0 as controls, different concentrations of resveratrol and different pretreatment times all affected the expression of CXCR3, IL4I1, and IDO genes in human umbilical cord mesenchymal stem cells. Regarding resveratrol concentration, the mRNA expression fluctuated at concentrations of 0.25, 1, 2.5, 10, and 25 μM without a clear pattern, but overall, the expression of mRNA in human umbilical cord mesenchymal stem cells was upregulated after resveratrol addition, and the expression of CXCR3, IL4I1, and IDO genes reached its highest point at a concentration of 2.5 μM. Regarding resveratrol pretreatment time, from 12, 24, 48, to 72 h, mRNA expression initially increased, reaching its maximum at 24 h, and then began to decline.

[0064] The figure shows that resveratrol pretreatment can increase the expression levels of CXCR3, IL4I1, and IDO genes in MSCs. Under the conditions of resveratrol concentration of 2.5 μM and pretreatment time of 24 h, the upregulation of CXCR3, IDO, and IL4I1 mRNA expression in MSCs was most significant. The results indicate that the gene expression levels of mesenchymal stem cells are better under the conditions of resveratrol concentration of 2.5 μM and pretreatment time of 24 h.

[0065] Example 3: Effects of resveratrol pretreatment on CXCR3 and IDO expression proteins in MSCs.

[0066] Experimental materials: human umbilical cord mesenchymal stem cells, PBS buffer, trypsin solution, IP lysis buffer, loading buffer.

[0067] Experimental method: After counting the resuspended cells from Example 1, the cells were divided into 3 × 10⁻⁶ cells per well. 5 10 cells, 2 mL in volume, were seeded into 6-well plates. After 12 hours of cell attachment, the original culture medium was removed, and the cells were divided into groups and given fresh culture medium.

[0068] The control group was given 2 mL of DMEM / F12 basal medium;

[0069] The experimental group was given 1 mL of DMEM / F12 basal medium containing 2.5 μM resveratrol.

[0070] Each group was set up with 3 replicates and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

[0071] Extracting total protein from human umbilical cord mesenchymal stem cells: Remove the original culture medium, wash 2-3 times with PBS buffer, and add 45 μL of IP lysis buffer (containing 1% PMSF) to each well.

[0072] Gently but thoroughly scrape the cells from the bottom of the culture well into the centrifuge tube using a cell scraper, ensuring all cells are scraped off and fully contacted with the lysis buffer.

[0073] Place centrifuge tubes containing cell lysates on ice and lyse for 40 minutes. During lysis, every 10 minutes, briefly remove the centrifuge tubes from the ice, vortex them for 10 seconds, and immediately return them to the ice.

[0074] After lysis for 40 minutes, the microcentrifuge tubes were equilibrated and placed in a centrifuge at 4°C. The tubes were centrifuged at 15000g for 10 minutes, and the supernatant was collected to obtain the total protein.

[0075] Total protein quantification was performed using the BCA method: First, 20 μL of protein standard was added to prepare a standard curve. The sample was diluted 20 times with ultrapure water, and the total volume was kept consistent with that of the standard. 200 μL of BCA working solution was added, and the sample was incubated at 37°C for 30 min. The absorbance at 562 nm was detected by an ELISA reader, and the total protein concentration was calculated using the corresponding standard curve.

[0076] The total protein of human umbilical cord mesenchymal stem cells was detected by Western blot (WB): the protein sample was added to loading buffer, heated in a metal bath at 100℃ for 10 min, and then loaded at a protein amount of 50 μg.

[0077] The parameters were set as follows: First stage stacking gel: 75V constant voltage, 30min;

[0078] Second stage separation gel: 125V constant voltage, 60min.

[0079] After 30 minutes of semi-drying, seal with 5% skim milk at room temperature for 2 hours.

[0080] Incubate with CXCR3 primary antibody (1:1000) overnight at 4°C, and wash the membrane three times with TBST for 10 min each time;

[0081] Incubate the membrane with HRP-labeled secondary antibody (1:10000) at room temperature for 2 h, then wash the membrane three times with TBST for 10 min each time.

[0082] Finally, the developer was prepared according to the ECL kit instructions, and the mixture was exposed and developed using a BIO-RAD developer. The grayscale ratio was then analyzed using ImageJ software.

[0083] Example 4: Effect of resveratrol pretreatment on IL4I1 expression in MSCs. The supernatant of MSCs culture medium after resveratrol pretreatment was collected for analysis.

[0084] Prepare the standard according to the instructions. Add 50 μL of standard and sample to each well, except for the blank well. Incubate at 37°C for 30 min and discard the liquid in the well.

[0085] After washing the plate 5 times with the prepared washing solution, add 50 μL of enzyme-labeled reagent (except for the blank wells), incubate at 37°C for 30 min, discard the liquid in the wells, wash the plate 5 times, add 50 μL of color development solution A and B in sequence, and develop the color at 37°C in the dark for 10 min.

[0086] After adding 50 μL of stop solution, the OD value was measured at a wavelength of 450 nm using an ELISA reader. The standard curve was then calculated, and the sample OD value was substituted into the standard curve to calculate the IL4I1 concentration.

[0087] The results obtained in Examples 3 and 4 are as follows Figure 3 As shown. Figure 3 This study reflects the effect of adding 2.5 μM resveratrol and pretreatment for 24 h on the total protein expression levels of CXCR3, IDO, and IL4I1 in human umbilical cord mesenchymal stem cells.

[0088] As shown in the figure, compared with the control group (MSCs) NC Compared to the MSCs pretreated with 2.5 μM resveratrol for 24 h, Res The relative expression levels of CXCR3 and IDO proteins in human umbilical cord mesenchymal stem cells (MSCs) were significantly increased, approaching 1.5 times that of the control group. Res IL4I1 protein expression levels in MSCs NC The concentration was below 6 pg / ml, rising to nearly 9 pg / ml. This indicates that pretreatment with 2.5 μM resveratrol for 24 h can promote the expression of CXCR3, IDO, and IL4I1 proteins in MSCs.

[0089] Experimental results show that resveratrol can increase the expression level of mesenchymal stem cell proteins.

[0090] Example 5: Effect of resveratrol pretreatment on the expression level of chemokine receptor CXCR3 on MSC cell membrane.

[0091] Experimental materials: human umbilical cord mesenchymal stem cells, DMEM / F12 basal culture medium, Zombie dye.

[0092] Experimental method: After counting the resuspended cells from Example 1, the cells were divided into 8 × 10⁸ cells per well. 4700 μL of cells were seeded into 24-well plates. After 12 hours of cell adhesion, the original culture medium was removed, and two groups were divided into two groups and given fresh culture medium.

[0093] The control group was given 700 μL of DMEM / F12 basal medium;

[0094] The experimental group was given 700 μL of DMEM / F12 basal medium containing 2.5 μM resveratrol.

[0095] Each group was set up with 5 replicates and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

[0096] Flow cytometry was used to detect the expression level of chemokine receptor CXCR3 on the membrane of human umbilical cord mesenchymal stem cells: the culture medium was removed, the cells were washed once with PBS, and 500 μL of trypsin solution was added for digestion for 2 min. Under an inverted microscope, most cells were observed to float up as round dots. An equal volume of DMEM / F12 complete culture medium was added to stop the trypsin digestion. The cell mixture was collected into a 1.5 mL EP tube, centrifuged, and the supernatant was discarded.

[0097] Resuspend in 1 mL of PBS solution, centrifuge at 4 °C and 500 g for 5 min, and discard the supernatant.

[0098] Then add 100 μL of Zombie dye (1:500), incubate at room temperature for 5 min, add 200 μL of PBS to terminate the staining reaction, centrifuge at 4℃ and 500g for 5 min, and discard the supernatant.

[0099] Add 100 μL of CXCR3-AF647 antibody (1:100) to each sample under light-protected conditions, stain at 4℃ for 30 min, add 200 μL of PBS solution to terminate the staining reaction, centrifuge at 4℃ and 500g for 5 min, and discard the supernatant.

[0100] Resuspend MSCs cells in 200 μL of PBS solution and perform analysis.

[0101] The results obtained in Example 5 are as follows Figure 4 As shown. Figure 4 The image shows the expression level of the chemokine receptor CXCR3 on the membrane of human umbilical cord mesenchymal stem cells. Compared with the control group (NC), the peak of the 2.5 μM resveratrol pretreatment group (Res) shifted to the right, indicating an increase in positive signal. The bar chart shows that the 2.5 μM resveratrol pretreatment group (MSCs) showed a higher expression level. Res CXCR3 + Values ​​and control group (MSCs) NCCompared to double, this indicates that resveratrol pretreatment significantly increased the expression level of chemokine receptor CXCR3 on the membrane of human umbilical cord mesenchymal stem cells.

[0102] Experimental results showed that resveratrol pretreatment could significantly enhance the expression level of chemokine receptor CXCR3 on the MSC cell membrane.

[0103] Example 6: Effect of resveratrol pretreatment on the migration of MSCs in response to chemokine CXCL10.

[0104] Experimental method: After counting the resuspended cells from Example 1, the cells were divided into 3 × 10⁻⁶ cells per well. 5 10 cells, 2 mL in volume, were seeded into 6-well plates. After 12 hours of cell adhesion, the original culture medium was removed, and two groups were added with fresh culture medium to form a control.

[0105] The control group was given 1 mL of DMEM / F12 basal medium;

[0106] The experimental group was given 1 mL of DMEM / F12 basal medium containing 2.5 μM resveratrol.

[0107] The cells were cultured for 24 hours in a cell culture incubator at 37°C and 5% CO2.

[0108] Transwell migration assay of human umbilical cord mesenchymal stem cells: Remove the culture medium, wash once with PBS solution, and digest with 600 μL trypsin solution for 2 min.

[0109] Under a microscope, most mesenchymal stem cells were observed to float up as round dots. An equal volume of DMEM / F12 complete culture medium was added to stop trypsin digestion. The cell mixture was collected into a centrifuge tube, centrifuged at 500g for 5 minutes, and the supernatant was discarded.

[0110] Resuspend MSCs cells in DMEM / F12 complete medium. After counting the MSCs cells, seed them into the upper chamber of an 8μm well Transwell 24-well plate at a density of 8000 cells per well (200μL). Once the cells have adhered, remove the original medium from the upper chamber.

[0111] Add DMEM / F12 basal medium to the chamber of a Transwell 24-well plate.

[0112] The lower chamber was divided into an experimental group and a control group. The experimental group was given DMEM / F12 complete medium containing CXCL10, while the control group was given DMEM / F12 complete medium without CXCL10. Each group was set with 5 replicates and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h.

[0113] Crystal violet staining of human umbilical cord mesenchymal stem cells: After the Transwell 24-well plate culture was completed, the cells were washed three times with PBS solution in the upper chamber and then fixed with 4% PFA (paraformaldehyde) solution at room temperature for 20 min.

[0114] The upper chamber of the Transwell was then washed three times with PBS to remove any residual PFA solution.

[0115] Upper chamber cells were stained with crystal violet for 15 minutes at room temperature.

[0116] After staining, the cells were washed with PBS to remove any unbound crystal violet dye. Unmigrated cells remaining in the upper cavity were gently wiped away with a cotton swab. The cells were then placed under a microscope for bright-field imaging and counted using Image J. Migrating cells stained purple appeared clearly as dark dots under bright-field microscopy.

[0117] The results obtained in Example 6 are as follows Figure 5 As shown. Figure 5 Microscopic images and bar charts showing the migration of human umbilical cord mesenchymal stem cells from the upper chamber to the lower chamber of the Transwell.

[0118] As shown in the figure, compared with the blank control group (Con), the number of human umbilical cord mesenchymal stem cells that migrated from the upper chamber to the lower chamber of the Transwell was greater in the resveratrol (Res) treatment group.

[0119] After the addition of chemokine CXCL10, the number of human umbilical cord mesenchymal stem cells migrating from the upper chamber to the lower chamber of Transwell increased, and the number in the resveratrol (Res) treatment group was significantly higher than that in the control group.

[0120] The experimental results showed that pretreatment with 2.5 μM resveratrol (Res) for 24 h significantly increased the number of human umbilical cord mesenchymal stem cells migrating from the upper chamber to the lower chamber of Transwell, and that 2.5 μM resveratrol treatment could enhance the responsiveness of human umbilical cord mesenchymal stem cells to high levels of CXCL10.

[0121] Example 7: Effects of resveratrol pretreatment of MSCs on inflammatory T cells

[0122] Experimental materials: mice, FACS solution, erythrocyte lysis buffer, Zombie live / dead dye

[0123] Experimental methods:

[0124] T cell isolation and culture: C57BL / 6N mice were euthanized, spleen tissue was removed, spleen tissue was ground with a filter screen, cell suspension was collected with FACS solution, centrifuged at 4℃ and 350g for 5 min, and supernatant was discarded.

[0125] Add 3 mL of red blood cell lysis buffer to resuspend the cells and incubate at room temperature for 5 min.

[0126] Add 6 mL of FACS solution to terminate the lysis, centrifuge at 4 °C and 350 g for 5 min, and discard the supernatant containing lysed red blood cell fragments and hemoglobin.

[0127] Resuspend cells in RPMI-1640 complete medium, and take 2.5 × 10⁻⁶ cells. 5 Add cells / well to a 24-well cell culture plate.

[0128] Establishing a co-culture system: Inoculating MSCs NC / MSC Res (8000 cells / well) were placed in the upper chamber of a 24-well Transwell plate; after culturing T cells derived from the spleen tissue of C57BL / 6N mice for 48 hours, the upper chamber of MSCs was transferred to the upper layer of the T cell plate wells and cultured for a total of 2 days.

[0129] Collect the co-cultured T cells, centrifuge at 4℃ and 500g for 5 minutes, and discard the supernatant.

[0130] Resuspend the cells in Zombie live / dead dye (1:500) and stain at room temperature in the dark for 5 min. Terminate the Zombie live / dead dye reaction, centrifuge at 500g for 5 min, and discard the supernatant.

[0131] Resuspend T cells in blocking solution (0.5 μL / tube), block at 4°C in the dark for 15 min, terminate, centrifuge, and discard the supernatant.

[0132] Resuspend the cells in a mixture of antibodies (1 μL CD3-FITC, 1 μL CD4-BV605), incubate at 4°C in the dark for 30 min, dilute with excess buffer to terminate the antibody binding reaction, centrifuge, precipitate, and discard the supernatant.

[0133] Fix the cells with a fixative at 4°C in the dark for 30 min, then stop the fixation, centrifuge, and discard the supernatant. Resuspend the cells in 200 μL LFACS solution (PBS + 2% serum), filter through a 200-mesh cell sieve, and then proceed with the analysis.

[0134] The results obtained in Example 7 are as follows Figure 6 As shown. Figure 6 The CD4 counts of the blank control group (NC), the mesenchymal stem cell group (MSC), and the resveratrol-treated mesenchymal stem cell group (MSC-Res) were displayed. + T cell activation levels.

[0135] Among them, the activation level of control group (NC) cells was greater than 80%, the activation level of mesenchymal stem cell group (MSC) cells was 60%, and the activation level of resveratrol-treated mesenchymal stem cell group (MSC-Res) cells was less than 40%.

[0136] The results showed that resveratrol treatment of human umbilical cord mesenchymal stem cells significantly inhibited CD4+. + The activation level of T cells has a positive impact on the regulation of inflammatory T cells by human umbilical cord mesenchymal stem cells.

[0137] Example 8: Transfection of MSCs with Red Fluorescent Protein (RFP)

[0138] Experimental materials: human umbilical cord mesenchymal stem cells, lentivirus containing the red fluorescent protein (RFP) gene

[0139] Experimental Method: After counting the resuspended cells from Example 1, the cells were seeded into T75 culture flasks. After 12 hours of cell adhesion, the original culture medium was removed, and DMEM / F12 basal medium containing lentivirus of the red fluorescent protein (RFP) gene was added at a concentration of 1×10⁶ cells / mL. 6 2 × 10 cells were added to the virus 7 The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 48 hours.

[0140] Remove the culture medium and replace it with DMEM / F12 complete medium containing 2.5 μg / mL puromycin. Continue culturing for 24 hours for screening.

[0141] The culture medium was removed, and two groups were added with fresh culture medium: one group was given basal culture medium as a control group, and the other group was given DMEM / F12 basal culture medium containing 2.5 μM resveratrol as an experimental group. Both groups were cultured at 37°C in a 5% CO2 cell incubator for 24 h. Two groups of human umbilical cord mesenchymal stem cells transfected with red fluorescent protein were obtained.

[0142] Example 9: Effect of resveratrol pretreatment on renal homing rate of MSCs in mice with lupus nephritis.

[0143] Experimental materials: Lupus nephritis mice

[0144] Experimental methods: Lupus nephritis mice were divided into two groups. Human umbilical cord mesenchymal stem cells transfected with red fluorescent protein, obtained in Example 8, were injected into each mouse via tail vein at a dose of 8 × 10⁸ cells. 5 1 cell, 200 μL in volume.

[0145] 24 hours later, mice were anesthetized with isoflurane and then sacrificed. The mice's kidneys were collected and washed with PBS buffer.

[0146] The fluorescence intensity was measured using an IVIS instrument (Caliper Life Sciences, Hopkinton, MA) with the following filter settings: excitation wavelength 594 nm and emission wavelength 647 nm.

[0147] The results obtained in Example 9 are as follows Figure 7 As shown.

[0148] Figure 7 The image shows a fluorescence diagram and a bar chart of fluorescence intensity of human umbilical cord mesenchymal stem cells in the kidneys of normal mice and lupus nephritis mice.

[0149] As can be seen from the figure, compared with the control group (MSCs) Con Compared to ), mesenchymal stem cell (MSCs) cultured in basal culture medium Con ) and mesenchymal stem cells (MSCs) cultured in resveratrol-containing medium Res Radiation efficiency was significantly increased in the kidneys of mice, and the resveratrol group (MSCs) Res The radiation efficiency of resveratrol was the highest, indicating that the addition of resveratrol can promote the homing rate of human umbilical cord mesenchymal stem cells in the kidneys of mice with lupus nephritis.

[0150] The results showed that pretreatment with 2.5 μM resveratrol for 24 h significantly increased the number of human umbilical cord mesenchymal stem cells homing in the kidneys of mice with lupus nephritis.

[0151] Example 10: Effects of resveratrol-pretreated MSCs on the renal immune microenvironment in mice with lupus nephritis.

[0152] Experimental materials: Lupus nephritis mice, human umbilical cord mesenchymal stem cells transfected with red fluorescent protein.

[0153] Experimental methods:

[0154] Lupus nephritis mice were divided into two groups. The two groups of human umbilical cord mesenchymal stem cells transfected with red fluorescent protein obtained in Example 8 were injected via tail vein, at a dose of 8 × 10⁸ per mouse. 5 One cell, 200 μL in volume, was slowly and evenly injected into mice with lupus nephritis.

[0155] Three days later, mice were anesthetized with isoflurane and euthanized. Their kidneys were collected, washed with PBS solution, and 7 mL of digestion solution was added. The mice were placed in a 37°C constant temperature shaker and shaken for 40 min. Digestion was terminated by adding 7 mL of RPMI-1640 complete culture medium.

[0156] Filter using a 70μm (200 mesh) filter; centrifuge the filtrate at 1500rpm for 5min at 4℃, carefully discard the supernatant, and avoid disturbing the precipitate.

[0157] After resuspending the cells in RPMI-1640 complete medium, centrifuge at 1500 rpm for 5 min at 4°C and discard the supernatant.

[0158] Red blood cell lysis: Resuspend cells in 2 mL of red blood cell lysis buffer and lyse at room temperature for 5 min; stop lysis by adding 2 mL of FACS solution, centrifuge at 1500 rpm for 5 min at 4 °C, and discard the supernatant.

[0159] Wash the cells with PBS buffer and transfer them to 1.5 mL centrifuge tubes. Centrifuge at 1500 rpm for 5 min at 4 °C, discard the supernatant, and resuspend the cells in 200 μL FACS solution.

[0160] T cell flow cytometry detection in mouse kidney: The kidney single cells collected by digestion were centrifuged at 4℃ and 500g for 5 min and the supernatant was discarded;

[0161] Resuspend the cells in Zombie live / dead dye (1:500), stain at room temperature in the dark for 5 min, then stop staining, centrifuge, and discard the supernatant.

[0162] Add blocking buffer (0.5 μL / tube) to resuspend cells, block at 4°C in the dark for 15 min, terminate, centrifuge, and discard supernatant;

[0163] Resuspend the cells in a mixture of antibodies (1 μL CD45-APC / fire 750, 1 μL CD4-FITC antibody / tube), stain at 4°C in the dark for 30 min; terminate the staining process, centrifuge, and discard the supernatant.

[0164] Fix the sample by drilling holes at 4℃ in the dark for 30 minutes, then stop the process, centrifuge, and discard the supernatant.

[0165] Resuspend cells in intracellular antibody (1 μL T-betPE / Cy7 antibody / tube), stain at 4°C in the dark for 30 min; terminate the treatment, centrifuge, and discard the supernatant;

[0166] The cells were resuspended and filtered through a 200-mesh cell sieve before being tested.

[0167] The results obtained in Example 10 are as follows Figure 8 As shown. Figure 8 The results of T cell infiltration in four groups of mice are provided, including:

[0168] Balb / c: A healthy wild-type mouse strain that serves as a normal control group and provides a baseline level of the renal immune microenvironment in a disease-free state.

[0169] MRL / lpr: This refers to the lupus model mouse group (disease control group), which showed significant lymphocyte infiltration and inflammation in the kidneys.

[0170] MRL / lpr+MSCs: Mesenchymal stem cell therapy group.

[0171] MRL / lpr+MSCs-Res: Resveratrol-treated mesenchymal stem cell therapy group.

[0172] CD45+ is a common antigen of all leukocytes, and the positive rate (CD45+ cells%) is the most direct indicator of the total immune cell infiltration level in tissues. The percentage of CD45+ cells in the MRL / lpr group was significantly higher than that in the Ballo / c healthy control group, confirming severe immune cell infiltration and inflammation in the kidneys of MRL / lpr model mice, consistent with the pathological characteristics of lupus nephritis. The percentage of CD45+ cells in the MRL / lpr+MSCs treatment group was significantly lower than that in the untreated MRL / lpr disease group, indicating that human umbilical cord mesenchymal stem cell therapy effectively reduced immune cell infiltration in the kidneys of lupus nephritis mice. Furthermore, the percentage of CD45+ cells in the MRL / lpr+MSCs-Res group was even lower than that in the MRL / lpr+MSCs group, suggesting that resveratrol-pretreated human umbilical cord mesenchymal stem cells may be more effective than ordinary MSCs in reducing renal immune cell infiltration.

[0173] Experimental results showed that pretreatment with 2.5 μM resveratrol for 24 h enhanced the inhibitory effect of MSCs on T cell infiltration in the kidneys of lupus nephritis mice and improved the renal immune microenvironment. The modified MSCs exerted a nephroprotective effect by significantly reducing total leukocyte infiltration in the kidneys and improving the disordered renal immune microenvironment in lupus nephritis.

[0174] In summary, this invention provides a traditional Chinese medicine monomer, resveratrol, as a culture medium additive. Pretreatment of human mesenchymal stem cells at a specific concentration (2.5 μM) and time (24 h) successfully enhanced the kidney homing rate of human mesenchymal stem cells in mice with lupus nephritis and improved the inhibitory effect of human mesenchymal stem cells on T cell infiltration in the kidneys of mice with lupus nephritis. This opens up a new avenue for the treatment of lupus nephritis and has significant clinical application and translational value in the field of autoimmune disease treatment.

[0175] The present invention has been described in the above-described embodiments; however, these embodiments are merely examples for implementing the present invention. It must be noted that the disclosed embodiments do not limit the scope of the present invention. Conversely, any modifications and refinements made without departing from the spirit and scope of the present invention are within the scope of patent protection of the present invention.

Claims

1. Application of a homing and drug-enhancing mesenchymal stem cell.

2. The application as described in claim 1, characterized in that, Human mesenchymal stem cells were seeded into a pretreated culture medium.

3. The application as described in claim 2, characterized in that, The seeding density of the human mesenchymal stem cells was 10. 5 ~10 7 cells / ml.

4. The application as described in claim 2, characterized in that, The pretreatment medium was based on DMEM / F12 medium.

5. The application as described in claim 2, characterized in that, The pretreatment culture medium contains resveratrol.

6. The application as described in claim 2, characterized in that, The final concentration of resveratrol in the pretreatment culture medium is 0.1-20 μM, preferably 2.5 μM.

7. The application as described in claim 2, characterized in that, The human mesenchymal stem cells were cultured for 24–72 hours.

8. The application as described in claim 5, characterized in that, The resveratrol mentioned above is used to improve the renal homing rate and immune microenvironment regulation capacity of mesenchymal stem cells in vivo.

9. The application as described in claim 2, characterized in that, The human mesenchymal stem cells are used to treat lupus nephritis and other types of nephritis.

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