转染BIG-3基因的人骨髓基质干细胞向软骨细胞诱导分化的实验研究  

Differentiation of human bone marrow stromal cells (hBMSCs) transfected with BIG-3 gene into chondrocytes

作　　者：戴洪山[1] 孟国林[1] 刘建[1] 马兴[1] 李立文[1] 张大伟[1] 李丹[1] 胡蕴玉[1] 

机构地区：[1]第四军医大学西京医院骨科研究所,西安710032

出　　处：《中华创伤骨科杂志》2006年第8期760-763,共4页Chinese Journal of Orthopaedic Trauma

基　　金：国家自然科学基金资助项目(30300356)

摘　　要：目的研究转染BIG-3基因对人骨髓基质干细胞(hBMSCs)向软骨纠胞诱导分化的影响。方法将构建好的pMSCVpuro-BIG-3转染至建系的hBMSCs,5μg/mL嘌罗霉素筛选12 d后,扩增并收集细胞,电泳证实特定条带无误。实验分三组：hBMSCs-BIG-3组、hBMSCs-EV组和hBMSCs组,每组重复6次,将三组细胞制成微粒模拟三维培养模式,分别加入浓度为400 ng/mL的重组人骨形态发生蛋白-2(rhBMP一2)作用14 d后,检测甲苯胺蓝及Ⅱ型胶原mRNA,利用MPS60病理图象分析系统采集图像,并进行灰度分析,半定量比较基质表达量。结果灰度值：hBMSCs-BIG-3组为684 481 822,hBMSCs-EV组为439 101 780,hBMSCs组为441 082 183,hBMSCs-BIG-3组灰度值明显高于后两组(P＜0．05),hBMSCs-EV组和hBMSCs组之间差异无显著性意义(P=0．862)。结论BIG-3基因有助于hBMSCs向软骨细胞诱导分化。Objective To investigate the differentiation of human bone marrow stromal cells （hBMSCs） transfected with BIG-3 gene into chondrocytes. Methods hBMSCs were stably transfected by pMSCVpuro-BIG-3 and screened for 12 days in a medium containing 5 μg/mL puromycin. The survival cells were amplified and collected. Polyacrylamide gel electrophoresis confirmed the existence of a special band. Experiments were carried out in three groups： hBMSCs-BIG-3, hBMSCs-EV and hBMSCs. The procedures were repeated six times in each group. Cells were cultured in the form of micromass to simulate three-dimensional culture. Induction medium contained IMDM, 5% FBS and 400 ng/mL rhBMP-2. After culture for 14 days, collagen Ⅱ mRNA expressions were detected in each group of cells by toluidine blue staining and hybridization in situ. The gray values of three groups were analyzed by MPS60 patho-image analysis system, and their matrix quantities were compared semiquantitatively. Results The gray values of the three groups were as follows： hBMSCs-BIG-3 684 481 822, hBMSCs-EV 439 101 780, hBMSCs 441 082 183. The matrix quantity of hBMSCs-BIG-3 group was the highest of the three groups （ P 〈 0. 05）. Conclusion Existence of BIG-3 gene is helpful for differentiation of hBMSCs into chondrocytes.

关 键 词：BIG-3基因 人骨髓基质干细胞 软骨诱导 重组人骨形态发生蛋白-2 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]