lncRNA GAS5调控Notch通路对骨髓间充质干细胞软骨分化的影响

Effect of lncRNA GAS5 on cartilage differentiation of BMSCs by regulating Notch pathway

目的观察长链非编码RNA(lncRNA)生长停滞特异性转录本5(GAS5)对骨髓间充质干细胞(BMSCs)软骨分化的影响,并探讨其机制是否与Notch通路有关。方法分离、培养大鼠BMSCs,并经倒相差显微镜观察细胞形态及流式细胞仪检测表面标志物表达鉴定证实。将第3代BMSCs分为对照组、p-EGFP-C1组(转染p-EGFP-C1空载体质粒)、p-EGFP-C1-lncRNAGAS5组(转染p-EGFP-C1-lncRNAGAS5过表达质粒)、p-EGFP-C1-lncRNAGAS5+DAPT组[转染p-EGFP-C1-lncRNAGAS5过表达质粒+100 ng/mLNotch信号通路特异性阻断剂2,4-二氨基-5-苯噻唑(DAPT)],采用qRT-PCR法检测各组细胞lncRNAGAS5表达。各组细胞进行软骨分化诱导,HE染色观察细胞形态及数量,免疫细胞化学法检测Ⅱ型胶原纤维α1(COL2a1)、蛋白聚糖(Aggrecan)阳性表达率,Westernblotting法检测Notch1和兔抗Split多毛增强子1(Hes-1)蛋白表达。结果p-EGFP-C1-lncRNAGAS5组、p-EGFP-C1-lncRNAGAS5+DAPT组细胞lncRNAGAS5相对表达量均明显高于对照组、p-EGFP-C1组(P均<0.05)。各组细胞均呈软骨细胞形态改变,即呈三角或多角形,细胞核为蓝紫色,细胞质和细胞外基质为红色,成团生长;p-EGFP-C1-lncRNAGAS5组与p-EGFP-C1-lncRNAGAS5+DAPT组软骨细胞数量明显多于对照组和p-EGFP-C1组,以p-EGFP-C1-lncRNAGAS5+DAPT组软骨细胞数量最多。与对照组及p-EGFP-C1组比较,p-EGFP-C1-lncRNAGAS5组及p-EGFP-C1-lncRNA GAS5+DAPT组细胞COL2a1、Aggrecan阳性表达率均升高,Notch1、Hes-1蛋白相对表达量均降低,且p-EGFP-C1-lncRNAGAS5+DAPT组变化更明显(P均<0.05)。结论lncRNAGAS5可通过抑制Notch通路激活来促进BMSCs的软骨分化。

Objective To observe the effect of long non-coding RNA(lncRNA)-growth arrest specific transcript 5(lncRNA GAS5)on cartilage differentiation of bone marrow mesenchymal stem cells(BMSCs),and to explore whether its mechanism is related to Notch pathway.Methods The rat BMSCs were isolated and cultured,the cell morphology was observed with inverted phase contrast microscope,and the cells were confirmed by detecting the expression of surface markers with flow cytometry.The third-generation BMSCs were divided into four groups:control group,p-EGFP-C1 group(transfected with p-EGFP-C1 empty vector plasmid),p-EGFP-C1-lncRNA GAS5 group(transfected with p-EGFP-C1-ln⁃cRNA GAS5 overexpression plasmid),and p-EGFP-C1-lncRNA GAS5+DAPT group[transfected with p-EGFP-C1-ln⁃cRNA GAS5+100 ng/mL Notch signaling pathway specific blocker 2,4-diamino-5-phenylthiazole(DAPT)].The qRT-PCR was used to detect the expression level of lncRNA GAS5 in BMSCs.The cartilage differentiation was induced in each group.HE staining was used to observe the morphology and number of BMSCs,immunocytochemical method was used to detect the positive expression rates of type II collagen fiberα1(COL2α1)and Aggrecan,and Western blotting was used detect the protein expression levels of Notch1 and hairy and enhancer of Split 1(Hes-1).Results The relative expres sion levels of lncRNA GAS5 in the p-EGFP-C1-lncRNA GAS5 group and p-EGFP-C1-lncRNA GAS5+DAPT group were significantly higher than those in the control group and p-EGFP-C1 group(all P<0.05).The cells in each group showed changes in the morphology of chondrocytes,which were triangular or polygonal,the nucleus was blue-purple,the cyto⁃plasm and extracellular matrix were red,and they grew in clusters;the numbers of chondrocytes in the p-EGFP-C1-ln⁃cRNA GAS5 group and p-EGFP-C1-lncRNA GAS5+DAPT group were significantly larger than those in the control group and p-EGFP-C1 group,with the largest number of chondrocytes in the p-EGFP-C1-lncRNA GAS5+DAPT group.Com⁃pared with the control group and p-EGFP-C1 group,the positive expression rates of COL2a1 and Aggrecan were significant⁃ly higher,the relative expression levels of Notch1 and Hes-1 were significantly lower in the p-EGFP-C1-lncRNA GAS5 group and the p-EGFP-C1-lncRNA GAS5+DAPT group,and the changes in the p-EGFP-C1-lncRNA GAS5+DAPT group were more obvious(all P<0.05).Conclusion LncRNA GAS5 can promote the cartilage differentiation of BMSCs via in⁃hibiting the activation of Notch pathway.Key words:lncRNA GAS5;Notch pathway;bone marrow mesenchymal stem cells;cartilage differentiation;Notch1;Hes-1 sion levels of lncRNA GAS5 in the p-EGFP-C1-lncRNA GAS5 group and p-EGFP-C1-lncRNA GAS5+DAPT group were significantly higher than those in the control group and p-EGFP-C1 group(all P<0.05).The cells in each group showed changes in the morphology of chondrocytes,which were triangular or polygonal,the nucleus was blue-purple,the cyto⁃plasm and extracellular matrix were red,and they grew in clusters;the numbers of chondrocytes in the p-EGFP-C1-ln⁃cRNA GAS5 group and p-EGFP-C1-lncRNA GAS5+DAPT group were significantly larger than those in the control group and p-EGFP-C1 group,with the largest number of chondrocytes in the p-EGFP-C1-lncRNA GAS5+DAPT group.Com⁃pared with the control group and p-EGFP-C1 group,the positive expression rates of COL2a1 and Aggrecan were significant⁃ly higher,the relative expression levels of Notch1 and Hes-1 were significantly lower in the p-EGFP-C1-lncRNA GAS5 group and the p-EGFP-C1-lncRNA GAS5+DAPT group,and the changes in the p-EGFP-C1-lncRNA GAS5+DAPT group were more obvious(all P<0.05).Conclusion LncRNA GAS5 can promote the cartilage differentiation of BMSCs via in⁃hibiting the activation of Notch pathway.