The Experimental Study Of Adipose-derived Stromal Cells For Treatment Of Stress Urinary Incontinence In Rats

Objective:ADSCs were purified through percoll density gradient centrifugation and preplate technique,and to differentiate rat ADSCs into myoblasts in vitro.Labeled ADSCs will be transplatated into the SUI rats around the posterior urethral.By urodynamics,morphological,transmission electron microscope and multiphoton microscopic imaging changes of urethra and its surrounding tissue.Our objective is to observe whether ADSCs transplant in the treatment of SUI rats is effective and feasible or not and its possible mechanism.Rat ADSCs transplantation can be provided a new way for cell transplantation therapy of stress urinary incontinence.Methods:1. ADSCs were purified through percoll density gradient centrifugation and preplate technique. ADSCs were further identified by flow cytometry technology for CD44,CD90, CD34,CD45,CD106 antibodies and by adipogenesis,osteogenesis cellular differentiation.2. A lentiviral vector with green fluorescent protein(GFP) was be used to transfection ADSCs.3. The twice passage ADSCs were treated for 24 h with 5-azacytidine (5-Aza, 10μmol-L-1) to induce cellular differentiation. The morphology and ultrastructure of treated ADSCs were observed by inverted microscope, transmission electron microscope and multiphoton microscopic imaging. The expression of desmin identified by immunohistochemistry, was used to identify myoblasts differentiation.The sarcomeric and Desmin expression were detected with RT-PCR and Western Blot.4. Bilateral pudendal nerve transaction was preparation of rat animal model of SUI. Simulated human body urodynamic method of the maximum bladder capacity of experimental animals (MBV), abdominal leak point pressure (ALPP), maximum urethral closure pressure (MUCP), and the functional length of the urethra (FUL);and HE staining, Masson special stain, Mallory staining and special nature of the urethral sphincter striated muscle ultrastructure in transmission electron microscopy studies confirmed the successful production of animal models of SUI.5. Rats were randomly assigned to transplant Group I (n=16) injected with ADSCs and control GroupⅡ(n=9) injected with culture medium without serum. Urodynamic testing, evaluation of dynamic changes in animal,morphology and ultrastructure of were observed by inverted microscope, transmission electron microscope and multiphoton microscopic imaging to evaluation the effective and feasible of ADSCs transplantation.Results:1. Method combining improved two-step enzymatic digestion with preplating technique is a kind of easy and practicable in some degree to obtain relative high purity of ADSCs.The purified ADSCs by limited-dilution appeared fibroblast-like shapes and proliferated rapidly in vitro. Results of flow cytometry showed that ADSCs were positive for CD44 and CD90, and negative for CD34, CD45 and CD106. Florescence intensity increased was companied with MOIs increased,but MOI of 8 fluorescence intensity was the strongest.82%-86%of successively passaged P3 ADSCs were GFP-positive.2. The morphology of ADSCs treated with 5-Aza under inverted microscope and multiphoton microscopic imaging changed from fusiform to polygon or astrocyte. With transmission electron microscope, the ultrastructure of the treated ADSCs was visible.The magnitude of cells was not identical, the surface of some cells had abundance of microvilli. Intracytoplasm had rich organelles, mitochondrium and rough endoplasmic reticulum.The induced ADSCs demonstrated positive staining for desmin by immunohistochemistry.The outcome of RT-PCR and Western Blot manifested that the gene expression of Desmin was up regulated in induced ADSCs.3.The urethral of female SD rat is a tubular structure about 2.0 cm. Its structure includes circular striated muscle layer,circular smooth muscle layer,longitudinal smooth muscle layer,dense connective tissue and epithelium from outside to inside. Bilateral pudendal nerve transection of the method can be successfully produced in rat model of SUI.Compared with those at baseline, the average quantity of maximum bladder volume (MBV) were increased by (0.89±0.04)ml (P<0.01); Abdomen pressure leak point pressure (ALPP) were significantly decreased by (15.46±5.49)cmH2O, respectively (P<0.01).Maximum urethra closure pressure (MUCP) and functional length of the urethra (FUL) were decreased by (7.97±3.25) cmH2O and (0.69±0.32)cm,respectively (P<0.01). Modeling of the posterior urethra and surrounding tissue morphology:sphincter loose arrangement of some muscles fracture phenomenon, muscle thinning; the proportion of connective tissue increase in sparse collagen fibers arranged in disorder.4. Two weeks after transplantation, by urodynamic testing, compared with the control group all parameters of MBV, ALPP, MUCP, FUL were statistically significant (P <0.01). By ADSCs transplantation,the urethral circular striated muscle layer can be significantly strengthened and thickened, sphincter morphology and function was significantly improved, the surrounding connective tissue close, supportive role to be strengthened. Striated muscle segmentation analysis revealed that ADSCs therapy for 4 weeks the ratio of muscle area and the length of the shaft were to be increased significantly. Collagen contents around the urethra and its surrounding was significantly higher compared with control group(P<0.01).Conclusion1. This part study manifests that high purify ADSCs can be obtained through digestion,percoll density gradient centrifugation and preplate technique. Lentiviral vector with green fluorescent protein(GFP) was be successfully used to transfection ADSCs, which creates good condition for long time observation of ADSCs in vivo.2. It was concluded that under specific induction setting, ADSCs can be induced to differentiate into myoblasts, providing a potential new option in stem cell transplantation therapy for SUI.3. In this part study,rats models of SUI were obtained by transaction of bilateral pudendal nerve,which established the foundation of extending experiment. The striated urethral sphincter of female rats was considered as a closed ring surrounding the urethral from the middle to the distal. It suggests that this may be the main site of urinary continence mechanism.4. ADSCs transplanted into rats with stress urinary incontinence can survive, divide and integrated into the host urethral sphincter,and it can be improved the control of urinary function. The possible mechanism of ADSCs may be adopted to differentiate into myoblasts, increased urinary sphincter system, and to improve urethral closure mechanism mucosal achieved. With the development of tissue engineering technology, it provided a new concept and method to treat stress urinary incontinence.