长链非编码RNA DANCR促进滑膜间充质干细胞向软骨细胞的增殖与分化

摘要

BACKGROUND: A number of studies have shown that long non-coding RNA DANCR can play an important role in various pathophysiological processes through Wnt/β-catenin signaling pathway.OBJECTIVE: To explore the effect of long non-coding RNA DANCR on the proliferation and chondrogenesis of synovium-derived mesenchymal stem cells.METHODS: Passage 3 synovium-derived mesenchymal stem cells were obtained and transfected with pcDNA3.1-GP (control) and pcDNA3.1-GP(DANCR Homo) (experimental). Cell viability was estimated at 1, 2, 3, 4, 5, 6 and 7 days after DANCR transfection. The passage 3 cells were cultured in the chondriogenic medium for 14 days. And the chondrogenesis potential of cells was examined by toluidine blue staining. The chondrogenic-specific marker genes Aggrecan, Type II collagen (Col2) and Sox9 were determined by Real-time PCR.RESULTS AND CONCLUSION: The synovium-derived mesenchymal stem cells exhibited "S"-shaped curves in the two groups, with cell arrest at 1-2 days and rapid proliferation beginning at 3 days. Cell counting kit-8 assay and toluidine blue staining showed overexpressing DANCR significantly promoted proliferation in synovium-derived mesenchymal stem cells. The aggregates from synovium-derived mesenchymal stem cells in the experimental group had a greater amount of toluidine blue staining than the control group. In addition, we detected the higher expression of chondrogenic specific marker genes, such as Col2, Sox9 and Aggrecan, in the experimental group than the control group at 14 days after chondrogenic induction (P < 0.05). These results demonstrate that long non-coding RNA DANCR could enhance chondrogenic differentiation and proliferation of human synovium-derived mesenchymal stem cells and increase the expression of chondrogenic specific marker genes.%背景:多项研究表明,长链非编码RNA DANCR可通过Wnt/β-catenin信号通路在多种病理生理过程中发挥重要作用.目的:探究长链非编码RNA DANCR在滑膜间充质干细胞向软骨细胞增殖与分化过程中的作用.方法:取第3代人滑膜间充质干细胞,分别以pcDNA3.1-GP质粒和pcDNA3.1-GP(DANCR Homo)质粒转染滑膜间充质干细胞,分别设为对照组和实验组,培养7 d内,检测两组细胞增殖曲线;培养14 d后,组织学观察两组细胞成软骨能力;qRT-PCR检测两组成软骨分化后细胞的软骨特异性基因表达.结果与结论:①细胞增殖曲线:两组细胞增殖曲线大致呈"S"型,一二天内细胞处于相对停滞期,从第3天开始细胞进入快速增殖时期,实验组细胞增殖速度明显快于对照组;②组织学观察:两组细胞外基质均呈蓝色,符合软骨细胞特征,实验组细胞外基质蓝染强于对照组;③qRT-PCR检测:成软骨诱导14 d后,实验组软骨特异性基因Ⅱ型胶原、蛋白多糖、Sox9基因表达显著高于对照组(P<0.05);④结果表明:长链非编码RNA DANCR可促进滑膜间充质干细胞向软骨细胞的增殖与分化,并加强软骨特异性基因的表达.