The Effect Of The Expression Of Loxl-1、Fibulin-5and Elastin In The Connective Tissue Around The Urethra In SUI Model Rat By TGF-β1Combined HUCMSCs Transplantation

Objective1To established the method of separating mesenchymal stem cells (MSCs) from thefull-term pregnant women umbilical cord,and the approach of proliferation invitro.To study the biologic characteristics and phenotypic characteristics of MSCs invitro,preparing for the application of MSCs in the following research.2Added TGF-β1to induce human umbilical cord mesenchymal stem cells todifferentiate into fibroblasts, laying the foundation for further experiments3To Simulate of human pregnancy, child birth, birth trauma, ovaryectomized to buildanimal models of SUI SD rat.TGF-β1combined hUCMSCs was injected around theurethra,according urodynamics, sneezing experiment, urethra and morphologicalchanges in the bladder and elastic fibers. To observe the expression of Loxl-1,Fibulin-5and Elastin on surrounding tissues of urinary bladder and and provide theevidence for the treatment of SUI.Methods1In the sterile condition,obtain the umbilical cord sample from healthy full-term fetus,useorganization adherent method to separate the umbilical cord mesenchymal stemcells. MSCs are cultured with10%fetal bovine serum in DMEM/F12medium.2Detect cell surface markers by flow cytometry; Observe the morphology of cells byoptical microscope; CCK8assays cell proliferative capacity and depicts the growthcurve; Induce the MSCs to differentiate into adipocytes and osteoblasts and to verifythe multiple differentiation capacity in vitro.3Prepare of cells seeded,experimental group added TGF-β1(5ng/ml), control groupnormal cultured for7days, identify induced cells the expression of vimentin and Icollagen by immunohistochemistry.4Establishment of SUI animal model:①experiment group: pregnant females SD rats are anaesthetized in2hours after birth. Fry’s14F catheter will be inserted into thevagina2-3cm. Inject5ml saline into the balloon to maintain this staus for4hours.2weeks later with ovariectomy, the rats are routinely breeding for1month. In this way,SUI model is built.②The positive control group: pregnant female SD rats areanaesthetized in2hours after birth, maintaining this status for4hours. The rats areroutinely reared for1month.③The negative control group: general breeding, notspecial treatment.In order to test the success of the rat model, all rats are used BL-420biological functional experimental system to test urodynamics, in addition to thepositive rate of sneeze experiment.5The SUI model rats were divided into three groups, one group was injected withsaline, a group of injection hUCMSCs, another group was injected with TGF-β1jointhUCMSCs, January underwent urodynamic testing and sneeze experiment afterroutinely breeding for1month.6Sacrificed the rats, draw the materials from urethra and surrounding tissue andbladder, by changes in HE and elastic fiber staining to test changes of morphologyand elastic fibers in the urethra and bladder tissue.Results1It began to climb out of the cells by tissue culture in7-12days, and get primary cellsafter3weeks. The cells are fusiform, fibroblast-like shaped morphology, the growthrate after is significantly faster passage, more uniform cell morphology.Cell growthcurve shows that cell growth from3days to enter the logarithmic growth phase.Thethird generation cell express CD29、CD44、CD90、CD105, but hematopoietic cellsurface markers CD45, endothelial cell marker CD31, and is closely related to graftrejection surface markers CD40and HLA-DR expression are very low. The culturedcells can be induced to become fat cells and osteoblasts In vitro experiments,mesenchymalstem cells can be induced to adipocytes and osteoblasts in vitro. All thisshows that the cultured cells are mesenchymal stem cells.2TGF-β1can induce hUCMSCs to fibroblasts and promote the expression of type Icollagen and vimentin.3Simulate human pregnancy and delivery, dystocia, birth trauma, low estrogen can establish a stable SUI rat model. The maximum bladder capacity of the experimentalrats, the positive control group and negative control group of rats were1.67±0.37ml,2.94±0.12ml and3.1±0.27ml; leak point pressure values were27.22±2.42mmHg,32.06±1.20mmHg and32.86±1.64mmHg. The experimentalgroup were significantly lower than the two 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(76.2%)was significantly higher than the twocontrol groups. HE staining of urethra and surrounding tissue and bladder:experimental group compared with the negative and the positive control group,urethral muscle layer was thinner, partly muscular appearance breakage; fasciaconnective tissue components increased elastic fibers loose, disorganized.Bladderneck elastic fiber staining was only visible obvious perivascular elastic fibers dyed.，and there was no significant difference between the experimental group and thecontrol group.4SUI rats injected hUCMSCs around the urethra model: the experimental group wasdivided into hUCMSCs, hUCMSCs joint TGF-β1and saline group. The leak pointpressure of hUCMSCs and hUCMSCsjoint TGF-β1transplantation group were30.63±1.49mmHg and31.05±2.13mmHg, maximum bladder capacity was2.93±0.26ml and2.94±0.31ml, both were significantly higher than the saline group, andthere was no significant difference before and after saline injections (P>0.05), nostatistically significant difference between hUCMSCs and hUCMSCs joint TGF-β1transplantation group (P>0.05), sneeze positive rate of two transplantation groupswas significantly lower than the saline injection group.HE stain morphology ofurethra and bladder and surrounding tissue: compared with saline injection group,hUCMSCs and hUCMSCs joint TGF-β1group compared with urethral muscularthickening of the muscle and connective tissue between closely. Visible muscularbladder neck tissue thickening and continuous. Elastic fiber staining saline urethra andsurrounding connective tissue elastic fibers small, low-density and irregular,hUCMSCs and hUCMSCs joint TGF-β1group significantly increased elastic fibersand arranged in neat rows. Bladdertissueelasticfiberstaining obvious. 5Immunohistochemistry detect Loxl-1, Fibulin-5and Elastin changes in urethra andsurrounding tissue and bladder, hUCMSCs and hUCMSCs joint TGF-β1expressionof above indexes were increased compared with the saline group. RT-PCR showedhUCMSCs joint TGF-β1can increase expression of Loxl-1, Fibulin-5and Elastin, thedifference was statistically significant.Conclusion1With adherent method can obtain relatively pure hUCMSCs, and purifiedhUCMSCs low expression CD31, CD45, CD40, HLA-DR, strong expression ofCD29, CD90, CD44, CD105. Cell has a good ability in vitro amplification and stabletraits. TGF-β1can induce hUCMSCs to fibroblasts and promote the expression oftype I collagen and vimentin.2According to the pathogenesis of SUI, the simulation of human pregnancy,childbirth, birth trauma, ovariectomized can be established relatively stable SUImodel. Injection hUCMSCs can improve the positive rate of urethra dynamicsindicators and sneezing experiments.HE staining and elastic fiber staining show thehUCMSCs injection in urethra and surrounding tissue can repair the urethra andbladder tissue structures.3For this experiment did not knockout genes to establish SUI mouse model,Exogenous TGF-β1joint hUCMSCs injection may promote elastic fiber-relatedcomponents Loxl-1, Fibulin-5expression and Elastin, and by repairing the defect andits associated proteins elastin and effective repair of pelvic connective tissue involvedin the structure and function of the pelvic floor the remodeling process, in order toimprove SUI symptoms.