骨髓间充质干细胞和丝素蛋白/壳聚糖支架构建组织工程尿液流出道的研究  

Study on the tissue-engineered urinary conduit constructed by bone marrow mesenchymal stem cells and silk fibroin/chitosan

作　　者：江伟 陈小刚[1] 汪前亮 Jiang Wei;Chen Xiaogang;Wang Qianliang(Department of Urology,Huangshi Central Hospital,Affiliated Hospital of Hubei Polytechnic University,Edong Healthcare Group,Huangshi 435000,China;Hubei Key Laboratory of Kidney Disease Pathogenesis and Intervention,Huangshi Central Hospital,Affiliated Hospital of Hubei Polytechnic University,Edong Healthcare Group,Huangshi 435000,China;Hubei Province Key Laboratory of Occupational Hazard Identification and Control,Wuhan University of Science and Technology,Wuhan 430065,China)

机构地区：[1]鄂东医疗集团黄石市中心医院(湖北理工学院附属医院)泌尿外科,435000 [2]鄂东医疗集团黄石市中心医院(湖北理工学院附属医院)肾脏疾病发生与干预湖北省重点实验室 [3]武汉科技大学职业危害识别与控制湖北省重点实验室,430065

出　　处：《中华损伤与修复杂志（电子版）》2021年第1期6-14,共9页Chinese Journal of Injury Repair and Wound Healing(Electronic Edition)

基　　金：湖北省自然科学基金青年项目(2019CFB390);湖北省卫生健康科研基金资助(WJ2019H157)。

摘　　要：目的探讨骨髓间充质干细胞(BMSC)和丝素蛋白/壳聚糖支架构建组织工程管状移植物在兔体内进行尿流改道的可行性和有效性。方法本研究共选取雄性SPF级新西兰大白兔36只。(1)取新西兰大白兔6只,行耳缘静脉麻醉后,提取BMSC和并进行体外培养,选取第3代BMSC,进行爬片,流式细胞仪检测细胞表面标志物;(2)另取6只新西兰大白兔,耳缘静脉麻醉后,取膀胱组织块,对尿路上皮细胞进行分离、培养及鉴定;(3)采用Transwell法,对BMSC悬液进行培养,采用免疫荧光检测诱导分化后BMSC是否向尿路上皮表型分化,同时采用蛋白质印迹法及聚合酶链式反应检测UPIa、角蛋白(CK)-18的表达情况;(4)制备丝素蛋白/壳聚糖支架,并对制备好的支架的形态、孔隙率进行评估;(5)将诱导分化14 d后的BMSC浓度调至4×106/mL,逐滴加入盖满整个丝素蛋白/壳聚糖支架材料,设为实验组,对照组为同样浓度的细胞悬液置于细胞培养瓶培养,取培养1、3、5、7、9 d复合物,扫描电镜观察、评估丝素蛋白/壳聚糖支架上细胞生长情况,使用噻唑蓝比色法检测细胞在支架上的增殖能力,并进行活细胞/死细胞测试;(6)取剩下的新西兰大白兔24只,随机分为实验组和对照组,每组各12只。麻醉后,分别将接种BMSC和未种BMSC的丝素蛋白/壳聚糖支架埋入实验组和对照组新西兰大白兔大网膜,网膜包裹促血管化2周后行苏木精-伊红染色,检测血管生成情况,行免疫组织化学染色观察细胞在支架表面生长情况;(7)对2组新西兰大白兔行尿流改道术,于术后1、2、4、8周进行取材,并分别行苏木精-伊红染色和免疫组织化学染色,术后10周利用静脉尿路造影检测流出道通畅情况。结果(1)分离出的BMSC呈圆形,培养3 d后以长梭状细胞形态为主;培养10 d,细胞融合度可达到90%,呈典型的纺锤样形态;获取第3代BMSC,流式细胞仪检测结果显示:约99.9%的细胞表面表达Objective To explore the feasibility and effectiveness of tissue engineering tubular grafts constructed of bone marrow mesenchymal stem cell(BMSC)and silk fibroin/chitosan scaffolds for urinary diversion in rabbits.Methods A total of 36 male SPF New Zealand white rabbits were selected for this study.(1)Six New Zealand white rabbits were taken,after the ear vein anesthesia,the BMSC was extract and cultured in vitro,the third generation BMSC was selected,performed slides,and detected cell surface markers by flow cytometry.(2)Another 6 New Zealand white rabbits were taken,after the ear-marginal vein anesthesia,the bladder tissue block was taken and the urothelial cells were isolated,cultured and identified.(3)The Transwell method was used to culture the BMSC suspension,the immunofluorescence method was used to detect whether the induced cells differentiated into the urothelial phenotype,and the Western blotting and polymerase chain reaction were used to detect UPIa and cytokeratin(CK)-18 expression.(4)The silk fibroin/chitosan scaffold was prepared and the morphology and porosity of the prepared scaffold was evaluated.(5)Adjusted the BMSC concentration to 4×106/mL after induction of differentiation for 14 days,added drop by drop to cover the whole silk fibroin/chitosan scaffold material,set as the experimental group,and placed the control group with the same concentration of cell suspension on the cells culture flasks,scanning electron microscopy was used to observe the complexes cultured for 1,3,5,7 and 9 days,the growth of the cells on the silk fibroin/chitosan scaffold was evaluated,and the thiazole blue colorimetric method was used to detect the proliferation ability of the cells on the scaffold,and performed live cell/dead cell test.(6)The remaining 24 New Zealand white rabbits were taken and randomly divided into experimental group and control group,with 12 rabbits in each group.After the anesthesia,BMSC and unseeded BMSC silk protein/shell polysaccharide stent were buried in the experimental group and contro

关 键 词：干细胞 组织工程 尿流改道术 壳聚糖 丝素蛋白 支架材料 

分 类 号：R318.08[医药卫生—生物医学工程]