| Objectives:Stress urinary incontinence(SUI)refers to the involuntary leakage of urine during increased abdominal pressure,caused by defects in the support structures of the urethra and subsequent dysfunction.None of the current treatment strategies cure stress urinary incontinence,but only relieve SUI.In recent years,the effectiveness of exosomes has been confirmed by more and more studies,and mesenchymal stem cellbased therapy has become a promising treatment strategy derived from mesenchymal stem cell in the treatment of diseases.However,there are not many research reports on human umbilical cord mesenchymal stem cell-derived exosomes in the treatment of stress urinary incontinence,so studying the effect of human umbilical cord mesenchymal stem cell-derived exosomes on SUI is of great significance to find alternative treatments for SUI.This study aims to elucidate the role and possible mechanism of human umbilical cord mesenchymal stem cell-derived exosomes on SUI by detecting the expression levels of lysyl oxidase(LOX)and Fibulin-5 in anterior vaginal tissue of SUI rats.Methods:1.A rat model of stress urinary incontinence(SUI)was established using vaginal dilation(VD).The validity and timeliness of the SUI model established by the VD method were tested through sneezing experiments and urodynamic tests,thereby determining the time range for subsequent research.2.Collagenase P was used to digest human umbilical cord tissue and culture mesenchymal stem cells.Cell imaging system inverts the biological microscope to observe the morphology of cells;Cell surface markers were detected by flow cytometry;In vitro,human umbilical cord mesenchymal stem cells were induced to differentiate into adipocytes and bone cells to identify their multidirectional differentiation ability.3.The exosomes from the culture supernatant of human umbilical cord mesenchymal stem cells were enriched and purified by ultrafiltration sequential ultrahigh speed centrifugation,and the membrane surface markers of the exosomes were detected by Western blot;The morphology of exosomes was observed under transmission electron microscope(TEM);NTA detects the particle size of the exosomes.4.Local transplantation of hUCMSC-Exos into the anterior vaginal wall tissue of rats in each group was performed at different time points for urodynamic testing.After testing,the anterior vaginal wall tissue of rats was removed for subsequent experiments.Urodynamic test results were used to evaluate the effect of hUCMSCExos on SUI.5.RT-qPCR and ELISA were used to detect the expression levels of LOX and Fibulin-5 in the anterior vaginal wall tissue to explore the mechanism of action of hUCMSC-Exos on SUI.6.SPSS 26.0 software was used to analyze the data.The experimental data were expressed using mean ± standard deviation(X ±S).The comparison between the two groups was conducted using independent sample t-test,and the comparison between the groups was conducted using analysis of variance(one-way ANOVA test).P<0.05 was defined as a statistically significant difference.Visualize the results using Graph Pad Prism 7.0.Results:1.A rat model of SUI was established by simulating human pregnancy and delivery using VD method.The sneeze test results showed that the positive rates of sneeze test were 100%,83%,83%,66.7%,and 50% on the 1st,7th,14 th,21st,and28 th days after vaginal dilation,respectively.While the control animals were all negative.The results of urodynamic tests showed that in the control group,the leakage point pressure(LPP)were 43.150±1.552 mm Hg,42.561±2.102 mm Hg,42.784±2.057 mm Hg,43.676±1.794 mm Hg,and 43.073 ± 1.362 mm Hg,respectively;The MBV was 0.525 ± 0.104 m L,0.552±0.098 m L,0.623±0.116 m L,0.590±0.142 m L,and 0.572± 0.100 m L,respectively.In the SUI rat model group,the LPP were23.684±0.415 mm Hg,23.722±0.906 mm Hg,24.079±1.519 mm Hg,25.411±1.977 mm Hg,and 27.458 ±2.085 mm Hg,respectively;The MBV was 0.276±0.032 m L,0.286±0.019 m L,0.289± 0.012 m L,0.294±0.010 m L,and 0.295±0.015 m L,respectively.Compared with the healthy rats,LPP and MBV of the SUI rats in 1-day,7-day,14-day,21-day,and 28-day groups significantly decreased(P<0.05).On the first day after VD modeling,LPP(23.684±0.415 mm Hg)and MBV(0.276±0.032 m L)reached their lowest values,and then gradually recovered.At 28 days after dilation,LPP and MBV did not return to normal levels.2.Human umbilical cord mesenchymal stem cells were isolated by enzymatic digestion.The cells began to adhere to the wall on the second day,and they could be subcultured for the first time on the fourth to seventh days.As the number of passages increased,the cells were gradually purified.At P3 generation,the cells were long spindle shaped,fibroblast like,and swirling.With passage,the morphology of cells tends to be highly uniform.The cells cultured to the third generation expressed high levels of CD29,CD90,and low levels of endothelial cell marker CD31 and hematopoietic cell surface marker CD45.In vitro experiments have verified that the cells cultured in this experiment can be induced into adipocytes and bone cells.Meet the minimum standards for defining human MSCs proposed by the International Society for Cell Therapy.3.The exosomes derived from human umbilical cord mesenchymal stem cells were enriched and purified using ultrafiltration sequential ultrahigh speed centrifugation.The exosomes were shaped like a bowl,with a particle size of 30-150 nm.The exosomes strongly expressed surface markers such as CD63,Alix,and CD9.Comply with the criteria for defining exosomes proposed by the International Vesicle Association.4.After the intervention of hUCMSCs-Exos,the results of urodynamic tests showed that in the 3-day,7-day,14-day,and 21-day groups,compared with the blank group,the LPP and MBV in the control group and the experimental group were significantly lower(control group: LPP: P<0.05;MBV: P<0.05;experimental group:LPP: P< 0.05,MBV: P<0.05),and compared with the control group,the LPP and MBV in the experimental group were significantly higher(LPP: P<0.05,MBV: P<0.05).5.Through RT-qPCR detection,the m RNA changes of LOX protein and FIBULIN-5 protein related to collagen metabolism in the anterior vaginal wall tissue:Compared with the healthy rat group,the above indicators in the hUCMSCs-Exos group increased.RT-qPCR results showed that hUCMSCs-Exos could increase the m RNA expression levels of LOX and Fibulin-5,with a statistically significant difference(P<0.05).Changes in the protein content of LOX and Fibulin-5 in the anterior vaginal wall tissue detected by ELISA: Compared with the control group,the ELISA results on the 3rd,7th,and 21 st days showed that the protein content of LOX and Fibulin-5 in the hUCMSCs-Exos group increased significantly.Only the ELISA test results on the 14 th day showed no statistical difference between the two groups.Conclusions:1.The stable rat model of stress urinary incontinence used in this experiment can be successfully established by vaginal dilatation method.2.The collagenase digestion method adopted in this experiment was able to successfully isolate human umbilical cord MSC.The purified hUCMSC can be obtained by applying the enzymatic digestion method,which have good expansion ability in vitro.3.The exosomes derived from hUCMSC could be enriched and purified by ultrafiltration sequential ultra-high speed centrifugation method.4.hUCMSCs-Exos transplanted into the anterior vaginal wall of the SUI rat model increased the leak point pressure and maximum bladder capacity of the rats.5.hUCMSCs-Exos transplantation in the anterior vaginal wall of the SUI rat model significantly increased the gene and protein expression levels of LOX and Fibulin-5 in the anterior vaginal wall tissue,significantly improving the collagen and elastic fibers of the urethral support structure and exerting a therapeutic effect. |
PDF Full Text Request