Notch3对促进胰腺星形细胞活化的基因表达及信号通路的影响

Effects of Notch3 on gene expression and signal pathway of pancreatic stellate cell activation

目的:分离培养小鼠胰腺星形细胞(PSCs),检测Notch3对促进PSCs活化的基因表达及信号通路的影响。方法:对小鼠PSCs进行分离培养及传代。采用免疫荧光染色检测活化的小鼠PSCs中α-SMA,fibronectin及collagen I的表达;细胞分组为空白对照组(MOCK组),阴性对照组(转染Notch3 siRNA negative control,NC组),Notch3 siRNA组(转染Notch3 siRNA,N3 siRNA组)及Notch3 siRNA-1组(转染Notch3 siRNA-1,N3 siRNA-1组),提取各组总RNA,测定RNA浓度及纯度后,送至安诺优达基因科技(北京)有限公司进行转录组测序。结果:免疫荧光结果显示,在活化的PSCs中α-SMA,fibronectin及collagen I都有明显的表达。测序结果分析表明,与NC组相比较,在N3 siRNA组与N3 siRNA-1组,α-SMA基因,collagen I基因,fibronectin基因及CTGF基因均表达下调,与胶原蛋白代谢过程相关的基因表达上调,正向调节胶原生物合成的基因表达下调,而负向调节胶原生物合成的基因表达上调,PCNA基因表达下调;在N3siRNA组与N3siRNA-1组,调节细胞聚集的基因表达下调;在细胞组分部分,细胞外基质的基因表达下调;抑制PSCs中Notch3的表达可对细胞粘附分子信号通路,MAPK信号通路及TGF-β信号通路的组成成员的基因表达产生影响。结论:抑制Notch3的表达可抑制PSCs的活化,降低细胞增殖能力,降低迁移聚集能力及ECM合成的能力;抑制Notch3的表达可对其他的信号如细胞粘附分子信号通路,MAPK信号通路及TGF-β信号通路产生影响。

Objective:The pancreatic stellate cells(PSCs)of mice were isolated and cultured and the effects of Notch3 siRNA on PSCs gene expression were detected.Methods:were PSCs of mice were isolated and cultured.The expressions ofα-SMA,fibonectin and collagen I in activated PSCs were detected by immunofluorescence.The PSCs were divided into four groups,blank control group(MOCK group),negative siRNA control group(NC group),Notch3 siRNA group(N3 siRNA group)and Notch3 siRNA-1 group(N3 siRNA-1 group).Cell treatment:the same transfection method was applied to transfect PSCs for 48h.Then total RNA was extracted from each group,and the concentration and purity of RNA was measured.The transcriptome sequencing and analysis were performed by ANOROAD Gene technology(Beijing)co.,LTD.Results:The immunofluorescence results showed thatα-SMA,fibonectin and collagen I were significantly expressed in activated PSCs.The analysis of the sequencing results showed that the gene expressions ofα-SMA,collagen I,fibronectin,CTGF and PCNA of PSCs were down-regulated in N3 siRNA group and N3 siRNA-1 group comparing with NC group.The genes involved in collagen metabolism were up-regulated,the gene expression of positive regulation of collagen biosynthesis was down-regulated,while that of negative regulation of collagen biosynthesis was up-regulated in N3 siRNA group and N3 siRNA-1 group comparing with NC group.The genes that regulated cell aggregation were down-regulated,and the genes that regulated extracellular matrix were down regulated in N3 siRNA group and N3 siRNA-1 group comparing with NC group.Inhibition of Notch3 expression in PSCs could affect the gene expressions of cell adhesion molecule signaling pathway,MAPK signaling pathway and TGF-βsignaling pathway.Conclusion:Inhibition of Notch3 expression can inhibit activation of PSCs,and reduce the ability of proliferation,migration and aggregation and ECM synthesis of PSCs.Inhibition of Notch3 expression may affect other signal pathways such as cell adhesion molecule signaling pathway,MAPK signaling pathway and TGF-βsignaling pathway,but its effects need further validation.