Experimental Study On The Management By Transplanting Mesenchymal Stem Cells Into SUI Rat

Objective:1.To set up the method of separating mesenchymal stem cells(MSCs) from the bone marrow of SD rat and full-term human umbilical cord,and the ways of serial subcultivation in vitro.To study that two MSCs' biological properties and phenotypic characteristics in vitro,and tag two MSCs,provides an ideal cell sources for follow-up experiment.2.Simulation of human pregnancy,birth injury,low estrogen after castration and the relationship with stress urinary incontinence(SUI),set up animal models of SUI,labeled cells will be injected into the rats around the posterior urethral.Based on urodynamics,the positive rate of sneeze test,morphological changes of urethra and its surrounding tissue,to observe whether mesenchymal stem cells transplant in the treatment of SUI rats is effective and feasible or not.Materials and methods:1.Establishment of SUI model:①experimental group:pregnant female SD rats is anaesthetized immediately after birth.Fry's 14F catheter will be inserted into the vagina 2～3 cm,and thread fixed in the Vagina mouth.Inject 5ml physiological saline into the balloon and maintain this status for 4 hours.Two weeks later with bilateral oophorectomy,the rats are routinely reared for two months.In this way,SUI model is built.②the positive control group:pregnant female SD rats is anaesthetized immediately after birth and thread fixed in the Vagina mouth,maintaining this status for 4 hours.The rats are routinely reared for two months.In order to test the success of the rat model,all rats are used BL-410 biological function experimental system to test urodynamics,in addition to the positive rate of sneeze experiment,.2.In sterile condition,remove the rat tibia and femur,and apply adherent method to separate and purify rat bone marrow mesenchymal stem cells(BMSCs).In sterile condition,obtain the umbilical cord samples from healthy full-term fetus and apply double enzymatic method to separate and purify umbilical cord mesenchymal stem cells(UCMSCs),Two MSCs are cultivated with 10%fetal bovine serum(FBS) in DMEM/F12 medium.3.Detect cell surface markers with flow cytometry and identify cultured cells.Through the observation of cell morphology,MTT method assays cells value-added,depicts the growth curve and observes the biological characteristics of MSCs in vitro.4.Take lipofectamine 2000 as carriers and tag the two MSCs with PEGFP-N1.The labeled cells will be injected into two groups of rats' urethra separately.And then use BL-410 biological function experimental system to test urodynamics,and detect the positive rate of sneeze experiment after routinely reared for two weeks.5.Necropsied the rats,draw the materials from their bladder,urethra and the surrounding tissue,observe the pathological changes of their bladder,urethra and the surrounding tissue after HE and Masson staining.Observe the availability of green fluorescent of the experimental groups' biopsy specimens under the fluorescence microscope,to test if the transplantation of mesenchymal stem cells successfully grow and add value in vivo.Results:1.Set up SUI modle:Set up a stable animal model successfully by simulation of human pregnancy,dystocia birth trauma,low estrogen after castration.The maximum bladder capacity of the experimental rats,the negative control group and positive control group of rats were(1.93±0.49) ml,(3.21±0.58 ) ml and(3.14±0.52) ml;leak point pressure values were(23.83±4.23) mmHg,(30.57±3.31) mmHg and(30.28±1.99) mmHg. The experimental group were significantly lower than the two control groups about the two Urodynamic index(p＜0.05),and there was no statistical difference between the two control groups(p＞0.05);the positive rate of Sneeze test(72.2%) was significantly higher than the two control groups(both 0).2.MSCs in vitro and biological characteristics of MSCs:Primary cultured BMSCs were adherent and round 24 hours after inoculation.Most of the cells were adherent and 72 hours later become fusiform-shaped after inoculation.The cells were passaged after 10～12 days.The proliferation became faster after passaging.The third and the fourth passage of BMSCs were morphologically homogeneous.97.68 percent of the cultured cells expressed CD29;2.76 percent expressed CD45,which were identified as BMSCs. Most of the primary cultured BMSCs were adherent for more than 24～48 hours after inoculation.With 80 percent of the cells fusion in about 14～16 days,cells were fusiform-shaped,parallel-like or whirlpool-like growth.The proliferation became faster after passaging.The third and the fourth passage of UCMSCs were morphologically homogeneous.99 percent of the cultured cells of the third passage expressed CD29 and CD44,but negative for markers of the hematopoietic lineage,including CD34 and CD45, which were identified as UCMSCs.Most of the two MSCs were in still and DNA presynthetic phase(G0/G1 phase),and only few were in mitotic phase and DNA synthetic phase.Labeling rate of green fluorescent protein marker pEGFP-N1 were (15.18±2.57)%and(15.68±1.03)%.3.MSCs transplantation around urethra of SUI model:Experiments were divided into 3 groups:BMSCs injection group,UCMSCs injection group and PS injection group.The maximum bladder capacity of the BMSCs injection group and the UCMSCs injection group were(3.06±0.77&3.22±0.77) ml;leak point pressure values were(28.60±3.98&30.62±2.76) mmHg.Both were significantly higher than pretherapy(p＜0.05), and had no statistical difference between the negative and positive control group(p＞0.05),but PS injection group had no statistical difference between before and after treatment(p＞0.05).The maximum bladder capacity and leak point pressure of the PS injection group were less than the BMSCs injection group and the UCMSCs injection group(p＜0.05),but two MSCs transplantation groups had no statistical difference(p＞0.05).Two MSCs transplantation groups(16.7%) were significantly lower than the PS injection group(83.3%) about the positive rate of sneeze test.4.Morphology by light microscopy of urethra and the surrounding tissues after HE,Masson stain:The PS injection group noted sphincter loose,some of the muscles collapse and decreased muscle layer.;vaginal epithelial atrophy,increased connective tissue content.Two MSCs transplantation groups showed muscle layer thicker,but still relatively disordered and loose.Compared with the control groups,normal connective tissue had no significant improvement in vaginal epithelial atrophy.The bladder tissue of each group had no significant difference in light microscopy of morphology.Smooth muscle bundles were intact and continuous,which showed no abnormal at stratified epithelium.5.Urethra and the surrounding tissue were observed under the fluorescence microscope:2 weeks after transplantation,the green fluorescence could be seen in two MSCs transplantion groups,and the rest of the control group was not showed the expression of fluorescence.It was confirmed that the transplanted cells can survive and increase in rats.Conclusions:1.Based on the pathogenesis of SUI,according to the difference of the urodynamic index and the positive rate of sneeze test,a stable animal model can be established.2.Purified BMSCs can be obtained by adherent method,and were negative for CD45, positive for CD29.Purified UCMSCs can be obtained by double enzymatic method, and negative for CD34,CD45,CD31,positive for CD29,CD44.Most of the two MSCs are in G0/G1 phase,and can proliferate in vitro.The cultured MSCs are uniform and stable,and can be used for tissue engineering,pEGFP-N1 plasmid can be used as the marker of mesenchymal stem cells,liposome-mediated transfection of plasmid is applicable to cells' short-term tag.3.The imperfection of urethral sphincter and its surrounding tissue of PS injected rats, shows the change of morphology and biochemistry of pelvic framework is one of the cause of SUI.vaginal epithelial atrophy.Low estrogen and estrogen receptor status of ovariectomized rat model is one of the factors that caused SUI.4.BMSCs and UCMSCs can be survived and proliferated in the urethral and the surrounding tissue of injuried rats,and improve the urodynamic index and the positive rate of sneeze test.Morphology shows renovation of the support structures around the urethra,but fail to improve the low levels of estrogen status in rats.