骨髓间充质干细胞、肌样细胞藻酸钙复合凝胶在压力性尿失禁大鼠尿道周围的成肌效应研究

The effects of the gel compound from bone marrow mesenchymal stem cells and muscle-like cells/calcium alginate on myoblast formation around urethra in rats of stress urinary incontinence

目的探索骨髓间充质干细胞（bone marrow mesenchymal stem cell, BMSC）藻酸钙复合凝胶与经5-氮杂胞苷诱导的肌样细胞藻酸钙复合凝胶在压力性尿失禁（stress urinary incontinence,，SUI）大鼠尿道周围的成肌效应。方法SD大鼠BMSC体外分离、培养、鉴定；用5-氮杂胞苷诱导生成肌样细胞；2％藻酸钠与1％氯化钙溶液以5：1体积比配制藻酸钙凝胶，分别与BMSC和肌样细胞复合用于微量注射。72只6周龄雌性SD大鼠建立SUI模型后分为BMSC凝胶组、肌样细胞凝胶组、单纯凝胶组和空白对照4组，每组再分为3小组，于膀胱颈尿道移行部黏膜下肌层注射相应的复合凝胶。4周、8周时取各组大鼠尿道横截面进行HE染色、荧光示踪照相以及结蛋白、α-横纹肌动蛋白（α-SMA）染色检查。结果获得的BMSC第3代细胞表面细胞因子抗体CD，。阳性率为89．4％、CD，。为3．3％、CD45为2．5％、CD。为46．0％，荧光示踪照相见第3代培养细胞表达强绿色荧光。BMSC凝胶组和肌样细胞凝胶组4周、8周时，凝胶边缘血管长入并逐渐增多，荧光示踪BMSC聚集于新生血管周围，肌样细胞呈长梭形样生长，结蛋白、α—SMA明显阳性表达。结论BMSC、肌样细胞藻酸钙复合凝胶在大鼠SUI实验模型的微环境中具有向肌细胞分化的潜力。BMSC直接体内微环境诱导分化与5-氮杂胞苷体外诱导形成肌样细胞后植人体内微环境继续分化，短期结果无明显差异。

Objective To explore the effects of myoblast formation around the urethra of stress uri- nary incontinence （SUI） rats after treated with bone marrow mesenchymal stem cells （BMSCs） or muscle-like cells/calcium alginate composite gel injection therapy. Methods Isolation, cultivation and identification of Sprague-Dawley rat bone marrow mesenehymal stem cell were performed. 5-azacytidine was introduced to induce muscle-like cells. Calcium alginate gel was initially prepared by 2% sodium alginate and 1% calcium chloride solution at a volume ratio of 5： 1. Compounds of stem cells or muscle-like ceils were mixed with gel, respectively, and were prepared for microinjection. SUI was produced in 72 6-week-old female Sprague-Dawley rats. The rats were then divided into 4 groups： Gel group, stem cell-gel group, muscle-like cell-gel group and mock control group. Each group was further divided into 3 groups. Submueosal injection of gel was performed at urethra and bladder neck. After preparation of cross sections of rat urinary tract at 4 weeks and 8 weeks after injection, HE staining, fluorescent tracing, staining of Desmin and α- skeletal muscle actin （α-SMA） were performed. OD values of positive rates were compared. Results At 4 weeks and 8 weeks after injection in stem cell-gel group and muscle-like cell-gel group, growth of blood vessels gradually increased at gel edge, BMSCs and muscle-like cells gathered around the new blood vessels observed by fluorescence tracer, muscle-like cells grew into elongated spindle-like cells. Desmin and ct-SMA staining were positive in these groups, and the OD values in the stem cell-gel group and muscle-like cell-gel group was significantly higher than that from the gel only group and control group, but no difference was found between stem cell-gel group and muscle-like cell-gel group. Conclusions Compound of BMSCs, muscle-like cells and calcium alginate composite gel has the potential to differentiate into muscle cells in the microenvironment of SUI rat model. In short term, the myoblast formation potential is the same whether the BMSCs was introduced into the micro-environment in vivo directly, or the BMSCs was implanted into micro- environment after the formation of the muscles cells induced by 5-azacytidine in vitro.