BMP-2/bFGF双基因转染的骨髓间充质干细胞复合生物陶瓷骨促进兔桡骨缺损修复  

Repair efficacy of bioceramic bone composited with BMP-2/bFGF double gene-transfected bone marrow stem cells on radial defects in rabbits

作　　者：韩操[1] 张丽娜[2] 颜南[3] 王正东[4] HAN Cao;ZHANG Lina;YAN Nan;WANG Zhengdong(Department of Hand Surgery,Central Hospital Affiliated to Shenyang Medical College,Shenyang 110024,China;Department of Critical Care Medicine,Central Hospital Affiliated to Shenyang Medical College,Shenyang 110024,China;Rehabilitation Teaching and Research Department,School of Medical Applied Technology,Shenyang Medical College,Shenyang 110034,China;Department of Anatomy,School of Basic Medical Science,Shenyang Medical College,Shenyang 110034,China)

机构地区：[1]沈阳医学院附属中心医院手外科,沈阳110024 [2]沈阳医学院附属中心医院重症医学科,沈阳110024 [3]沈阳医学院医学应用技术学院康复教研室,沈阳110034 [4]沈阳医学院基础医学院解剖教研室,沈阳110034

出　　处：《中国医科大学学报》2022年第2期125-130,135,共7页Journal of China Medical University

基　　金：辽宁省自然科学基金(2019-MS-308)。

摘　　要：目的探讨骨形态发生蛋白2(BMP-2)/碱性成纤维细胞生长因子(bFGF)双基因转染的骨髓间充质干细胞(BMSC)复合生物陶瓷骨在兔桡骨缺损模型中的治疗作用,及其与EphB4/ephrin-B2信号通路的调控关系。方法体外分离培养兔BMSC并经流式细胞术鉴定,采用慢病毒载体将BMP-2/bFGF基因转染到兔BMSC,通过Western blotting鉴定过表达。制备BMSC复合生物陶瓷骨,植入兔挠骨缺损模型后,8周后取出缺损侧挠骨组织。HE染色观察桡骨组织形态变化;实时定量PCR检测成骨标志物Runt相关转录因子2(Runx2)、碱性磷酸酶(ALP)、骨桥蛋白(OPN)和Ⅰ型胶原(ColⅠ)的表达;采用Lane-Sandhu组织学评分分析桡骨修复情况;采用免疫荧光检测桡骨组织ColⅠ、OPN的表达;采用Western blotting检测桡骨组织中EphB4、ephrin-B2水平。结果BMP-2/bFGF双基因转染的BMSC复合生物陶瓷骨能够改善缺损桡骨组织形态,提高桡骨组织Lane-Sandhu组织学评分,上调桡骨组织中Runx2、ALP、OPN和ColⅠ的表达,并能够上调缺损桡骨组织中EphB4和ephrin-B2的水平。结论BMP-2/b FGF双基因转染的BMSC复合生物陶瓷骨对兔桡骨缺损有修复作用,其机制可能与激活EphB4/ephrin-B2信号通路有关。Objective To investigate the therapeutic effects of bioceramic bone composited with bone morphogenetic protein-2(BMP-2)/basic fibroblast growth factor(bFGF)-transfected bone marrow stem cells(BMSC)in a rabbit radial bone defect model and its relationship with EphB4/ephrin-B2 signaling pathway.Methods BMSCs were isolated and cultured in vitro and identified by flow cytometry.Then BMP-2 and bFGF genes were transfected into rabbit BMSCs using lentiviral vectors.Successful transfection was confirmed by Western blotting.Morphological changes in radial bone tissue were observed by HE staining.Expression of Runt-related transcription factor-2(Runx2),alkaline phosphatase(ALP),osteopontin(OPN),and collagen Ⅰ(ColⅠ)were assessed by real-time quantitative PCR.Radial bone repair was analyzed using Lane-Sandhu histological score.Expression of ColⅠ and OPN in radial bone tissue were detected by immunofluorescence.EphB4 and ephrin-B2 levels were detected by Western blotting.Results The bioceramic bone composited with BMP-2/bFGF double gene-transfected BMSCs improved the morphology of defective radial bone tissue,increased Lane-Sandhu histological scores in radial bone tissue,increased expression of Runx2,ALP,OPN,and ColⅠ in radial bone tissue,and up-regulated EphB4 and ephrin-B2 levels in defective bone tissue.Conclusion The bioceramic bone composited with BMP-2/bFGF double gene-transfected BMSCs elicit a better repairing effect on rabbit radial defects,potentially through activating the EphB4/ephrin-B2 signaling pathway.

关 键 词：骨形态发生蛋白2 碱性成纤维细胞生长因子 骨髓间充质干细胞 桡骨缺损 生物陶瓷骨 EphB4/ephrin-B2信号通路 

分 类 号：R318.08[医药卫生—生物医学工程]