稳定敲低人脐带间充质干细胞EPCR基因对其向成纤维细胞分化能力的影响

Effect of knocking down EPCR in hUC-MSCs on differentiation to fibroblasts

目的:构建稳定敲低人内皮细胞蛋白C受体(endothelial cell protein C receptor,EPCR)的人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs),检测敲低EPCR对hUC-MSCs向成纤维细胞分化能力的影响。方法:根据EPCR基因m RNA序列,合成3条小干扰RNA(small interfering RNA,siRNA)导入hUC-MSCs,通过检测EPCR蛋白表达筛选干扰效果最佳的siRNA。根据最佳siRNA序列构建慢病毒载体,包装重组慢病毒,感染hUC-MSCs,Real-time PCR和Western blot验证敲低效率。体外构建转化生长因子-β1(transforming growth factor beta1,TGF-β1)诱导hUC-MSCs向成纤维细胞分化模型,Real-time PCR和Western blot检测诱导前后α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和Ⅰ型胶原(typeⅠcollagen,ColⅠ)表达水平,以反映诱导效果。结果:以EPCR最佳siRNA序列构建EPCR基因RNAi慢病毒载体,成功敲低hUC-MSCs内EPCR的mRNA(t=28.86,P=0.000)和蛋白(t=88.60,P=0.000)表达水平。EPCR敲低后,hUC-MSCs合成的COL1A1和COL1A2在m RNA(P=0.044,P=0.000)和蛋白(P=0.000,P=0.000)水平减少。体外TGF-β1诱导可增加hUC-MSCs内α-SMA(F=369.713,P=0.000;F=451.060,P=0.000)、COL1A1(F=42.051,P=0.000;F=759.041,P=0.000)及COL1A2(F=359.205,P=0.000;F=764.348,P=0.000)在mRNA和蛋白水平的表达,促进其向成纤维细胞分化。但敲低EPCR,α-SMA(P=0.002,P=0.002)、COL1A1(P=0.002,P=0.000)及COL1A2(P=0.000,P=0.000)在mRNA和蛋白的升高水平明显降低。结论:成功获得EPCR稳定敲低的hUC-MSCs,证实敲低EPCR可减少hUC-MSCs内ColⅠ表达,并减弱其向成纤维细胞分化的能力。

Objective:To construct a lentiviral vector that is capable of knocking down EPCR in hUC-MSCs and to explore the effect of knocking down EPCR on differentiative capacity of hUC-MSCs into fibroblasts. Methods:Three siRNAs were synthesized according to the m RNA sequence of human EPCR and were transferred into hUC-MSCs. Western blot was conducted to analyze the suppression efficiency of three siRNAs to screen for an optimum siRNA. Then the shRNA targeting the optimum siRNA sequence was synthesized,cloned into lentiviral vector and packaged by lentivirus. HUCMSCs were infected with the recombinant lentivirus. The efficien cy of knocking down was detected both at mRNA and protein levels. HUC-MSCs were stimulated with TGF-β1. The expression levels of α-SMA and Col Ⅰ were detected by Realtime PCR and Western blot for evaluation of differentiative capacity of hUC-MSCs into fibroblasts. Results:Lentiviral vector that targeting to the optimal siRNA sequence of EPCR and can down-regulate the mRNA(t=28.86,P=0.000) and protein(t=88.60,P=0.000) expression of EPCR was constructed successfully. And knocking down of EPCR in hUC-MSCs decreased the mRNA(P=0.044,P=0.000) and protein(P=0.000,P=0.000) expression of COL1 A1 and COL1 A2. TGF-β1 could induce up-expression of α-SMA(F=369.713,P=0.000;F=451.060,P=0.000),COL1 A1(F=42.051,P=0.000;F=759.041,P=0.000) and COL1 A2(F =359.205,P =0.000;F=764.348,P=0.000) in mRNA and protein level and transform hUC-MSCs into fibroblasts. However,knocking down of EPCR in hUC-MSCs significantly decreased the expression of α-SMA(P=0.002,P=0.002) and COL1 A1(P=0.002,P=0.000) and COL1 A2(P=0.000,P=0.000) induced by TGF-β1 compared with that of control group. Conclusion:Knocking down of EPCR in hUC-MSCs are successfully constructed and could decrease the expression of Col Ⅰ as well as suppress the differentiative capacity of hUC-MSCs into fibroblasts.