摘要
[目的]探讨慢病毒介导转化生长因子β1(tansforming growth factor bata 1,TGF-β1)基因转染大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)诱导成软骨细胞分化的作用。[方法]密度梯度离心法分离培养BMSCs,探索病毒感染最佳条件,取第3代细胞,分为实验组和对照组,通过慢病毒载体将外源性TGF-β1基因转染入细胞,观察绿色荧光表达情况,Real-time PCR法和western blot法检测TGF-β1基因的mRNA和蛋白表达情况,7、14 d时行Ⅱ型胶原免疫细胞化学染色、甲苯胺蓝染色和Real-time PCR检测Ⅱ型胶原及聚集蛋白聚糖的表达。[结果]TGF-β1基因转染BMSCs后能够稳定表达。转染7、14 d时,实验组免疫细胞化学染色、甲苯胺蓝染色均为阳性。转染7、14 d时,实验组II型胶原mRNA均显著高于对照组(P<0.01)。转染7 d时,实验组聚集蛋白聚糖mRNA变化不明显,而14 d时显著高于对照组(P<0.01)。[结论]慢病毒介导的TGF-β1基因可成功转染大鼠BMSCs,并诱导其向软骨细胞分化。在此过程中软骨特异性标志Ⅱ型胶原和聚集蛋白聚糖的mRNA表达具有时间差异性。
[Objective] To investigate the effect of lentiviral-mediated transfer of transforming growth factor-beta1（ TGF-β1） gene on chondrogenesis of rat bone marrow mesenchymal stem cells（ BMSCs）. [Methods] Rat BMSCs were isolated by density gradient centrifugation method and cultured. The best infection conditions were determined,and the 3rd-passage BMSCs were divided into the treatment group and control group. TGF-β1 gene was transfected into the BMSCs using the lentiviral vector,and green fluorescent protein（ GFP） expression was observed to evaluate gene delivery efficiency. TGF-β1gene expression in BMSCs was detected by real-time PCR and western blotting. Immunocytochemical staining,toluidine blue staining,and real-time PCR were used to detect the expression of type II collagen and aggrecan at 7 and 14 days after gene transfection. [Results] TGF-β1 gene was stably expressed after transfection into BMSCs. Immunocytochemical and toluidine blue staining revealed the treatment group cells to be positive for type II collagen. At 7 and 14 days,mRNA expression of type II collagen was significantly higher in the treatment group than in the control group（ P〈0. 01）. At 7 days after transfection,aggrecan mRNA expression in the treatment group cells varied only slightly from that in the control cells. In contrast,at 14 days after transfection,aggrecan mRNA expression in the treatment group was significantly higher than that in the control group（ P 〈0. 01）. [Conclusion] TGF-β1 gene was successfully transfected into rat BMSCs using the lentiviral vector,and after transfection,the gene induced chondrogenesis of the BMSCs. Furthermore,there was a considerable time gap between the expression of type II collagen mRNA and aggrecan mRNA.
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2015年第7期637-643,共7页
Orthopedic Journal of China
基金
国家自然科学基金资助项目(编号:81373669)
浙江省重点科技创新团队计划资助项目(编号:2011R50022-01)
浙江省重点实验室计划资助项目(编号:2013E10024)
关键词
骨髓间充质干细胞
成软骨分化
转化生长因子β1
慢病毒载体
大鼠
bone marrow mesenchymal stem cells
chondrogenesis
transforming growth factor beta1
lentiviral vector
rat
作者简介
陈祁青，主治医师，博士研究生，研究方向：骨与关节疾病的临床与基础研究，软骨组织工程，（电话）13282412616，（电子信箱）13282412616@163．com
通讯作者：（电话）0571-86613684，（电子信箱）tongpeijian@163．com
