肝星状细胞中Notch1的表达及其意义  

The expression and role of Notch1 in hepatic stellate cells

作　　者：郑建建[1] 陈必成[1] 周振旭[2] 董培红[3] 俞富军[3] 

机构地区：[1]温州医科大学附属第一医院外科实验室,浙江温州325000 [2]温州医科大学附属第一医院腔镜外科,浙江温州325000 [3]温州医科大学附属第一医院感染内科,浙江温州325000

出　　处：《中国卫生检验杂志》2015年第2期168-172,共5页Chinese Journal of Health Laboratory Technology

基　　金：国家自然科学基金(81000176/H0317;81100292/H03-17);浙江省自然科学基金(Y2090326;Y2110634);王宝恩肝纤维化研究基金(20100002;20120127);温州市科技局资助项目(Y20110033;Y20120127)

摘　　要：目的研究Notch通路中Notch1在大鼠肝星状细胞(HSC-T6)的表达及其意义。方法细胞免疫组化检测细胞内Notch1的表达并采用MTT、real-time PCR及Western blot等多种技术检测HSC-T6细胞在Notch1沉默后增殖、凋亡和细胞外基质的变化;并利用细胞免疫荧光染色检测α-SMA的表达。结果 Notch1蛋白表达于HSC-T6细胞的胞质并随着HSC活化而增加。与正常组相比,TGF-β1组的细胞表现出Notch通路的激活,即Notch1及其下游分子Hes1的mRNA和蛋白表达量均表达上调;随着Notch1的沉默,MTT结果提示Notch1沉默组的细胞增殖率明显下降,但Western blot结果提示凋亡相关蛋白Caspase3表达量却未改变;同时细胞免疫荧光结果显示α-SMA表达下调且伴有Ⅰ型胶原及Notch下游分子Hes1表达下调,而E-cadherin表达量则明显上调。结论沉默Notch1可抑制TGF-β诱导的肝星状细胞活化,提示Notch1可促进肝纤维化进程。Objective To investigate the expression and role of Notch1 in HSC- T6 cells. Methods The expression of Notch1 protein was detected in hepatic stellate cells（ HSCs） by immunohistochemistry. The effects of Notch1 by Notch1 siRNA in TGF- β1- induced HSC- T6 cells were measured by MTT,real- time PCR and Western blot,and the rate of cell proliferation,apoptosis,and extracellular matrix were detected in TGF- β1- induced HSC- T6 cells transfected with Notch1 siRNA.Immunofluorescent analysis was performed to detect the expression of α- SMA. Results The result of immunohistochemistry showed that the expressions of Notch1 protein were existed in cytoplasm of HSCs and increased in TGF- β1 group. Compared with the control group,the mRNA and protein expressions of Notch1 and Hes1 which is a downstream gene of Notch signal pathway in TGF- β1- treated cells were highly increased,indicating that the Notch pathway was activated in cells after the treatment of TGF- β1. Next,HSC- T6 cells were transfected with Notch1 siRNA. Compared with the TGF- β1 group,the rate of cell proliferation rate was inhibited a lot in cells transfected with Notch1 siRNA. But the expression of Caspase3,a cell apoptosis- related protein,was not changed. Besides,the mRNA and protein expressions of type Ⅰ collagen and α- SMA were decreased after the transfection of Notch1 siRNA whereas the mRNA and protein expression of E- cadherin was increased. The result of immunofluorescent analysis confirmed the reduction of α- SMA. It also showed that Hes1 expression was significantly down- regulated after the transfection of Notch1 siRNA. Conclusion The activated HSCs by TGF- β were suppressed by the knockdown of Notch1,indicating that the progression of liver fibrosis could be promoted by Notch1.

关 键 词：肝纤维化 NOTCH1 Α-平滑肌肌动蛋白 

分 类 号：R575.2[医药卫生—消化系统]