沉默CTGF对肝星状细胞凋亡及胞外基质表达的影响

Effects of silencing CTGF on apoptosis and expression of extracelluar matrix proteins in rat hepatic stellate T6 cells

目的 观测沉默结缔组织生长因子(connective tissue growth factor, CTGF)基因对肝星状细胞T6(hepatic stellate cell T6,HSC_T6)的生长与凋亡,以及增殖细胞核抗原(Proliferating cell nuclear antigen, PCNA)、CTGF、整合素α5(integrin alpha 5,Itgα5)和Ⅰ型胶原(collagenⅠ, ColⅠ)表达的影响。方法 将CTGF-shRNA病毒感染HSC_T6(实验组)。空病毒感染的HSC_T6(空病毒组)或未感染的HSC_T6(正常组)作对照。在显微镜下观测被感染HSC_T6的生长与绿色荧光蛋白(green fluorescent protein, GFP)的表达变化;采用CCK-8(cell count kit-8)比色检测被感染HSC_T6的增殖情况;流式细胞仪(flow cytometry, FCM)检测被感染HSC_T6的凋亡情况。定量PCR、Western blot及免疫荧光细胞化学分别检测PCNA、CTGF、Itgα5和ColⅠ的表达变化。结果 在显微镜下观察到被感染的HSC_T6高表达GFP。CCK-8结果表明,与对照相比,沉默CTGF基因的HSC_T6生长显著受抑,72 h开始其差异具统计学意义(P<0.05)。FCM检测结果显示,沉默CTGF基因可诱导HSC_T6凋亡。定量PCR、Western blot及免疫荧光细胞化学结果证实,沉默CTGF基因可在mRNA及蛋白水平明显下调HSC_T6的PCNA、CTGF、Itgα5与ColⅠ的表达,与对照比较,差异均具统计学意义(P<0.05)。结论 沉默CTGF基因可下调HSC_T6的PCNA、CTGF、Itgα5及ColⅠ的表达,抑制HSC_T6的生长,诱导HSC_T6凋亡,其机制可能和HSC_T6的CTGF/Itgα5信号通路受阻紧密相关。

Objective To investigate the effects of silencing connective tissue growth factor(CTGF)gene on growth,apoptosis,and expression of proliferating cell nuclear antigen(PCNA),CTGF,integrin alpha 5(Itgα5)and collagenⅠ(ColⅠ)in rat hepatic stellate cell line T6(HSC_T6).Methods The CTGF shRNA lentiviral vectors with high infectivity were transfected into HSC_T6 cells,and the cells transfected with empty virus or not transfected served as control cells.Fluorescence inverted microscopy was used to observe the cell growth and the expression of green fluorescent protein(GFP)in the transfected HSC_T6 cells.CCK-8 assay and flow cytometry were employed respectively to detect the cell viability and apoptosis.The expression changes in PCNA,CTGF,Itgα5 and ColⅠwere measured by real-time PCR,Western blotting and immunofluorescence assay.Results Microscopic observation displayed that transfection with CTGF shRNA lentiviral vectors resulted in high expression of GFP in HSC_T6 cells.CCK-8 assay showed that silencing CTGF induced inhibited growth in the transfected cells,with significant difference since 72 h after transfection(P<0.05).Flow cytometry indicated that transfection of CTGF shRNA lentivirus led to the apoptosis of HSC_T6 cells.The results of real-time PCR,Western blotting and immunofluorescence assay confirmed that CTGF shRNA lentivirus effectively down-regulated the expression of PCNA,CTGF,Itgα5 and ColⅠ,with notable differences when compared the expression in control cells(P<0.05).Conclusion Silencing CTGF can down-regulate the expression of CTGF,Itgα5 and ColⅠ,inhibit the growth and induce the apoptosis of HSC_T6 cells,which might be closely associated with its interference for the CTGF/Itgα5 signaling pathway.