Myogenic Differentiation Of Human Umbilical Cord Blood Mesenchymal Stem Cells In Vitro And Its Effect On Improving Intrinsic Sphincter Deficiency

Stress urinary incontinence (SUI) is a common problem in woman. It will decent the patients' life quality and damage their physical and mental health. The incidence rate of the disease lifts with age increasing. The main reasons of SUI are concluded into two: the first one is over moving of the urinary tract and the second one is intrinsic sphincter deficiency (ISD). Though the methods of treatment are plentiful at present, it is difficult to solve the problem of ISD.In our study, based on the potential of myoblast differentiation, we transfected a eukaryotic expression plasmid vector pEGFP-Myocl labeled with enhance green fluorescent protein (EGFP) into MSCs isolated from umbilical cord blood and induced the cells differentiating into myoblast. After proliferative culture, the differentiated MSCs were grafted into the area surrounding the intrinsic sphincter of the model rat of SUI. Distribution and roles of MSCs-differentiated myoblast were investigated in the following tests.The main results are as follows.1. We establish the method of isolation and culture of mesenchymal stem cells (MSCs) from human umbilical cord blood (UB). The cell cycle was observed and theirs immunophenotypes were analyzed by flow cytometry. These cells were able to be purified and expanded by three passages and expressed CD29, CD44and CD105, but no hematopoietic lineage markers, such as CD34 and CD45. The experiment shows that non-hematopoietic stem cells can be isolated from umbilical cord blood and they have the same immunophenotypes with bone marrow-derived MSCs.2. Plasmid vector pEGFP-Myocl expressing in eukaryotic cells was constructed in success. According to the template of pAdApt. Myocl, we did PCR amplification and purified with electrophoresis. The product was cut by Kpn I and Age I and purified with electrophoresis, then ligated with linearized vector pEGFP-N1 in present of a ligase T4 DNA. The obtained pEGFP-Myocl was then transferred into an engineered bacterium JM109 to replicate the target plasmid. The fragment sequence was confirmed to be consistent with the reported one in Gene Bank.3. The plasmid pEGFP-Myocl was transfected into UB-MSCs by 15.3% with aid of lipotransfection method. After G418 screening, the expression of Myocardin was detected in the transfected MSCs with RT-PCR. Fluorescence was observed in the transfected cells under a fluorescent, indicating the existence of reporter gene. Immunohistochemical methods indicated the expressions of Myocardin and myosin. But they are negative in control. All of these indicated that in vitro cultured UB-MSCs were induced to differentiate into myoblast under the regulation of Myocardin.4. Rat model for Stress urinary incontinence can be obtained by simulating birth trauma and ovariectomy. Urodynamics measurement shows cystometry and modified leak point pressure of model rats significantly decreased compared with that in the control. HE strain shows the smooth muscle surround the intrinsic sphincter is atrophy and the fiber has fragmentation.5. MSCs-differentiated myoblasts were implanted into the area surrounding the intrinsic sphincter of the model rat of SUI. Sections at different time points were observed pathologically and immunostraining. We found that the planted cells were alive in the injection area in a short period and express some protein correlated with muscle, such as Desmin. Urodynamics measurement showed cystomety and modified leak point pressure of model rats significantly improved. But if the improving effect was the result of planted cells' regeneration and repair of muscle or just some kind of padding effect is not clear now.To sum up, the plasmid vector pEGFP-Myocl was successfully constructed in this experiment and transferred into UB-MSCs to induce them differentiating into myoblasts under the regulation of Myocardin. We also observed the differentiation and distribution after transplanting the cell into the area surrounding the intrinsic sphincter. Effect of myoblasts in the healing of SUI was explored by the urodynamics study. This study provides a new approach for theoretical instruction and potential clinical application in the treatment of SUI.