过表达骨形态发生蛋白4对兔骨髓间充质干细胞增殖与成软骨的影响  

The Effects of Bone Morphogenetic Protein 4 Overexpression on Proliferation and Chondrogenesis of Rabbit Bone Marrow Stem Cells

作　　者：张飞 党源[1] 薛来恩 温福利[1] 郑和平[1] 胡德庆[2] ZHANG Fei;DANG Yuan;XUE Laien;WEN Fuli;ZHENG Heping;HU Deqing(Fuzhou General Teaching Hospital of Bengbu Medical College,The 900th Hospital of The Joint Logistics Team,Fuzhou 350025,China;Department of Orthopedics,The First Affili ated Hospital of Fujian Medical University,Fuzhou 350004,China)

机构地区：[1]蚌埠医学院福总教学医院(联勤保障部队第九OO医院),福州350025 [2]福建医科大学附属第一医院骨科,福州350004

出　　处：《实验动物与比较医学》2020年第2期87-94,共8页Laboratory Animal and Comparative Medicine

基　　金：福建省自然科学基金(2017J05122)。

摘　　要：目的通过腺病毒(Ad)载体感染过表达骨形态发生蛋白4(BMP-4),研究BMP-4对兔骨髓间充质干细胞(BMSCs)体外增殖和成软骨的影响。方法全骨髓贴壁法分离获取BMSCs,传代培养提升细胞纯度,通过细胞形态学及流式细胞术测定表面分子标志物以鉴定细胞;设立病毒感染复数(MOI)梯度组,通过倒置相差显微镜和流式细胞仪确定最佳MOI;酶联免疫吸附试验(ELISA)及蛋白印迹法分析过表达BMP-4对细胞分泌Ⅱ型胶原(COLLⅡ),性别决定基因盒9(Sox9)和BMP-4的影响;细胞计数试剂盒8(CCK-8)法鉴定过表达BMP-4对BMSCs体外增殖的影响。结果原代细胞呈圆形、椭圆和梭形不等,第2代细胞呈典型成纤维样长梭形,漩涡状分布,低表达造血干细胞表面分子标志物CD34(1.94%)和CD45(2.13%),高表达CD44(98.2%)、CD29(99.7%)和CD90(98.8%)。MOI为100时,病毒感染效率较高,且对细胞损伤最小,为最适MOI。与对照组相比,过表达BMP-4能显著促进BMSCs的增殖,并能分泌成软骨特异指标COLLⅡ、BMP-4及Sox9(P<0.05)。结论过表达BMP-4能显著促进BMSCs的增殖及向软骨分化。Objective To investigate the effects of bone morphogenetic protein 4(BMP-4)overexpression on the proliferation and chondrogenesis of rabbit bone marrow stem cells(BMSCs)infected by adenovirus.Methods The BMSCs were isolated and cultured by whole bone marrow adherence method,the purity of BMSCs was improved by subculture,the cells were identified by cell morphology and surface molecular marker by flow cytometry,and the optimal multiplicity of infection(MOI)of adenovirus was detected by inverted phase contrast microscopy and flow cytometry.The effects of BMP-4 overexpression on the secretion of typeⅡcollagen(COLLⅡ),sry-related high mobility group-box gene 9(Sox9)and BMP-4 in chondrocytes were analyzed by enzyme linked immunosorbent assay(ELISA)and Western blotting.The effect of BMP-4 overexpression on the proliferation of BMSCs in vitro was detcted by cell counting kit-8(CCK-8).Results Primary cells were round,elliptical and spindle-shaped.The second generation cells were typical fibroblast-like long spindle-shaped,vortex-like distribution,with low expression of hematopoietic stem cell surface molecular markers CD34(1.94%)and CD45(2.13%),and high expression of CD44(98.2%),CD29(99.7%)and CD90(98.8%).MOI for 100,with higher infection efficiency and smallest damage to cells,is the most appropriate MOI.Compared with the control group,the overexpression of BMP-4 could significantly promote the proliferation of BMSCs and secrete a large number of COLLⅡ、Sox9 and BMP-4 proteins(P<0.05).Conclusion Overexpression of BMP-4 can significantly promote the proliferation and differentiation of BMSCs into chondrocytes in rabbit.

关 键 词：骨髓间充质干细胞 骨形态发生蛋白4 细胞增殖 成软骨 

分 类 号：R-33[医药卫生] Q95-33[生物学—动物学]