骨髓间充质干细胞在膀胱脱细胞基质上的生长及分化

背景:传统方法多采用平滑肌细胞和移行上皮细胞构建组织工程膀胱,并进行支架材料的双面种植,但由于平滑肌细胞取材培养困难,且体外传代有限,双面种植较为困难.目的:实验拟验证以骨髓间充质干细胞及膀胱脱细胞基质构建组织工程膀胱的可行性.设计:基础实验研究.单位:中山大学附属第二医院林百欣医学研究中心.材料:实验于2006-03/2007-05在中山大学附属第二医院林百欣医学研究中心完成.实验室级别为卫生部部属医院开放实验室.1月龄SD大鼠,雌雄不限,体质量80～100g,由中山大学实验动物中心提供.新鲜猪膀胱取自南方医科大学动物实验中心.方法:采用全骨髓培养连续贴壁法体外培养大鼠骨髓间充质干细胞,并应用流式细胞仪检测其表面抗原.采用去污剂洗涤法制备猪膀胱脱细胞基质,并测定其纯度及特性.将第3代骨髓间充质干细胞接种到膀胱脱细胞基质上,以添加25 ng/L血管内皮生长因子(VEGF165)的培养液进行体外、体内复合培养,检测其相容性.体内实验以细胞单独培养为对照,体外实验以未植入细胞的材料为对照,并模拟合适的微环境诱导骨髓间充质干细胞生长分化,分别在4,8周后取出动物体内的复合材料行组织切片检查,并进行免疫组织化学角蛋白染色,检测上皮细胞再生情况.主要观察指标:骨髓间充质干细胞与膀胱脱细胞基质的生物相容性.结果:①采用全骨髓法成功培养出骨髓间充质干细胞,行流式细胞仪检测细胞表面抗原显示,传代第3代细胞CD29阳性细胞为99.43%.②制备的膀胱脱细胞基质具有良好的生物特性,镜下见均质状态的基质和细丝状的胶原纤维.体内外相容性实验表明骨髓间充质干细胞与膀胱脱细胞基质具有良好的相容性,细胞生长状态良好.③4周后组织切片检查可见组织中较多炎症细胞浸润,胶原及弹性纤维排列紧密,免疫组织化学角蛋白染色见有薄层、不连续的单层上皮生长,8周后可见组织中已无明显炎症细胞浸润反应,胶原及弹性纤维排列紧密,免疫组织化学角蛋白染色见有薄层、连续的多层上皮生长.结论:骨髓间充质干细胞与膀胱脱细胞基质具有良好的生物相容性,并且在体内与周围组织有良好的生物相容性,可以作为构建组织工程膀胱的材料.

Culture and differentiation of bone marrow mesenchymal stem cells on bladder acellular matrix

BACKGROUND:Smooth muscle cells and transitional epithelial cells were traditionally used to construct tissue-engineered bladder and to perform double-sided implantation of scaffold.However,double-sided implantation is difficult to perform,because smooth muscle cells are difficult to isolate or culture in vitro and passage is limited.OBJECTIVE:To verify the feasibility of tissue-engineered bladder reconstruction with bone marrow mesenchymal stem cells(BMSCs)and bladder acellular matrix(BAM).DESIGN:A basic empirical study.SETTING:Linbaixin Medical Research Center,Second Affiliated Hospital,Sun Yat-sen University.MATERIALS:Experiments were performed at the Linbaixin Medical Research Center,Second Affiliated Hospital,Sun Yat-sen University from March 2006 to Mav 2007.The laboratory was the Opening Laboratory of Hospital Affiliated to Health Department of China.One-month old SD rats of either sex,weighting 80-100 g were provided by Animal Experimental Center of Sun Yat-sen University.Fresh porcine bladders were offered by Animal Experimental Center of Southern Medical University.METHODS:Whole bone marrow culture and successive adherence method was used to culture rat BMSCs in vitro.Flow cytometry was employed to detect surface antigen.Eradicator washing method was applied to prepare porcine BAM and measure its purity and characteristies.Third passage of BMSCs were inoculated in BAM and cultured in a medium containing vascular endothelial growth factor(VEGF165)(25 ng/L)in vive and in vitro to test compatibility.Cells cultured alone were considered to be controls for the in vivo trial,and materials non-implanted with cells were considered to be controls for in vitro trial.Suitable microenvironment was simulated to induce the differentiation of BMSCs.Four weeks and eight weeks later,compound materials were respectively removed to perform tissue section test.Simultaneously,immunohistochemistry keratin staining was conducted to examine regeneration of epithelial cells.MAIN OUTCOME MEASURE:Biocompatibility of BMSCs and BAM.RESULTS:①BMSCs were cultured by whole bone marrow method.Flow cytometry demonstrated that third passage of cells were positive for CD29(99.43%).②BAM had good biological characteristics.Homogen matrix and byssoid collagen appeared under a microscope.Compatibility trials showed good compatibility of BMSCs and BAM and well-growth cells.③Four weeks later,histological section test confirmed inflammatory cell infiltration,closely-arranged collagen and elastic fiber.Immunohistochemistry keratin staining showed lamellar and discontinuous simple epithelium.Eight weeks later,no inflammatory cell infiltration was found,and closely-arranged collagen and elastic fiber were detected.Immunohistochemistry keratin staining showed lamellar and continuous multiple epitheliums.CONCLUSIoN:With good compatibility,BMSCs and BAM appear to be an ideal material for bladder tissue engineering.