摘要
目的:观察右归饮含药血清对大鼠骨髓间充质干细胞(BMSCs)成软骨分化的效应,分析此过程中的micro RNA表达谱变化,探讨右归饮含药血清促进BMSCs成软骨分化的分子机制。方法:体外培养大鼠BMSCs,经TGF-β1基因慢病毒转染后48h给开始右归饮含药血清干预,培养7d,采用Ⅱ型胶原免疫组化染色、甲苯胺蓝染色、Real time PCR方法检测Ⅱ型胶原、聚集蛋白聚糖的表达。采用micro RNA芯片技术和生物信息学方法,分析差异micro RNA表达谱变化,预测关键micro RNA、信号通路及靶基因。结果:TGF-β1基因成功转染BMSCs并能稳定表达。转染组和右归饮组的甲苯胺蓝染色及Ⅱ型胶原免疫组化染色均呈阳性,空载组染色均为阴性;与空载组比较,转染组和右归饮组Ⅱ型胶原m RNA、聚集蛋白聚糖m RNA的表达上调(P<0.01);与转染组比较,右归饮组Ⅱ型胶原m RNA、聚集蛋白聚糖m RNA的表达上调(P<0.01)。右归饮组与转染组相比,17个micro RNA个显著上调,2个micro RNA显著下调(P<0.05,|fold change︱≥0.585)。mi R-24-3p、MAPK信号通路及MAPK13、MAPK14、TRAF6、MAP3K7基因被预测为关键micro RNA、信号通路及靶基因。结论:右归饮含药血清能较好地促进BMSCs成软骨分化,右归饮通过上调mi R-24-3p阻断MAPK信号通路可能发挥了重要作用。
Objective： To observe promoting effect of Youguiyin （YGY） medicated serum in chondrogenesis differentiation of bone marrow mesenchymal stem cells （BMSCs） and analyze on expression profile of microRNA, and investigate molecular mechanism of promoting effects of YGY medicated serum in chondrogenesis differentiation of BMSCs. Methods： Rat BMSCs were isolated and cultured in vitro. After TGF-β1 gene was transfected into the BMSCs through lentiviral vector, the BMSCs were administered with the YGY medicated serum for 7days. Immunocytochemical staining, toluidine blue staining, and Real-time PCR were used to detect the expression of type Ⅱ collagen and aggrecan, microRNA microarray and bioinformatics methods were used to test the microRNA expression profile changes in BMSCs and estimate the key microRNA, key signaling pathways and target gene. Results： TGF-β1 gene was successfully transfected into BMSCs and was stably expressed. The immunocytochemical and toluidine blue staining of cells in transfection group and YGY group were positive, while the staining ofcontrol lentiviral group was negative. Compared with the control lentiviral group, the mRNA expressions of type II collagen and aggrecan of cells in transfection group and YGY group were increased （P〈0.01）. Compared with transfection group, the mRNA expressions of type Ⅱ collagen and aggrecan of ceils in YGY group were increased （P〈0.01）. Compared with the transfection group, 17 microRNAs were significantly up-regulated and 2 microRNAs were significantly down-regulated in YGY group （P〈0.05, fold change （≥0.585）. MiR-24-3p, MAPK signal pathway, MAPK13, MAPK14, TRAF6 and MAP3K7 were predicted as the key microRNA, signal pathway and target genes. Conclusion： YGY medicated serum could effectively facilitate the differentiation of BMSCs into chondrocytes, which might be related to miR-24-3p up-regulation and MAPK signal pathway blocking.
出处
《中华中医药杂志》
CAS
CSCD
北大核心
2015年第5期1380-1386,共7页
China Journal of Traditional Chinese Medicine and Pharmacy
基金
国家自然科学基金(No.81373669)
浙江省重点科技创新团队计划资助项目(No.2011R50022-01)
浙江省重点实验室计划资助项目(No.2013E10024)~~
