抑制细胞因子RhoA的表达对肝星状细胞株HSC-T6胶原分泌的影响

Inhibition of RhoA expression reduces the secretion of extracellular matrix components in hepatic stellate cells

目的:观察并比较两种方法抑制RhoA的表达对细胞外基质成分,如Ⅰ型胶原、透明质酸以及层粘连蛋白合成的影响,为肝纤维化的基因治疗寻求新的途径.方法:设计并合成针对RhoA相同靶点的反义寡核苷酸和小干扰RNA,分别转染肝星状细胞株HSC-T6,逆转录PCR技术检测细胞中RhoA和Ⅰ型胶原mRNA的表达;Western blot检测细胞中RhoA蛋白质的表达;ELISA检测培养上清中透明质酸(HA)、层黏蛋白(LN)及PIIIP的含量.结果:转染RhoA反义寡核苷酸和小干扰RNARat1质粒后,HSC-T6中RhoA mRNA的表达分别由0.892±0.051、0.937±0.044降为0.113±0.024、0.212±0.042;蛋白质表达水平均明显下调;细胞内Ⅰ型胶原mRNA的表达分别由0.709±0.067、0.695±0.087降为0.436±0.037、0.201±0.044;细胞外基质成分如HA、LN及PIIIP的表达水平明显下降.两者相比,小干扰RNA具有更强的生物学效应.结论:靶向抑制细胞因子RhoA的表达可明显减少肝星状细胞株HSC-T6细胞外基质的合成,且小干扰RNA的效果明显优于针对同一靶点的反义寡核苷酸,显示RNAi技术应用于肝纤维化基因治疗的良好前景.

AIM：To investigate the effects of antisense oligodeoxynucleotide-and small-interfering RNA （siRNA）-mediated inhibition of RhoA expression on the secretion of extracellular matrix components in hepatic stellate cells （HSC-T6） to seek new approaches for the treatment of liver fibrosis.METHODS：An antisense oligodeoxynucleotide and a siRNA targeting the RhoA gene were designed and transfected into HSC-T6 cells.The expression of RhoA and type I collagen （Col I） mRNAs in HSC-T6 cells was detected by RTPCR.The expression of RhoA protein in HSC-T6 cells was detected by Western blot.The contents of hyaluronic acid,laminin and PIIIP in cell su-pernatants were measured by ELISA.RESULTS：After antisense oligodeoxynucleotide or siRNA transfection,the relative expression levels of RhoA mRNA were changed from 0.892 ± 0.051 and 0.937 ± 0.044 to 0.113 ± 0.024 and 0.212 ± 0.042,respectively;the expression of RhoA protein and the contents of extracellular matrix components decreased markedly;and the relative expression levels of Col I mRNA decreased from 0.709 ± 0.067 and 0.695 ± 0.087 to 0.436 ± 0.037 and 0.201 ± 0.044,respectively.CONCLUSION：Specific blockade of RhoA expression decreases the secretion of extracellular matrix components in HSC-T6 cells.RhoA-specific siRNA is superior to antisense oligodeoxynucleotide in down-regulating RhoA expression and therefore represents a better strategy for gene therapy of liver fibrosis.