摘要
目的探讨siRNA干扰大鼠盘状结构域受体2（discoidin domain receptor 2，DDR2）基因对肝星状细胞（hepatic stellate cell，HSC）的影响，评估DDR2在肝纤维化发生中的作用。方法（1）化学合成3对针对DDR2基因的siRNAs，转染肝星状细胞株（HSC—T6），筛选抑制效率最高的siRNA用于干扰实验；（2）将肝星状细胞株HSC-T6分为3组：正常组（normal，N组）、阴性对照组（negative control，NC组）和干扰组（siRNA—DDR2，SI组）。将筛选的抑制效率最高的siRNA以脂质体法转染HSC-T6细胞株，以lit—PCR检测其DDR2，α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）和I型胶原（collagen-I）的mRNA表达，Western blot检测DDR2蛋白表达变化；同时四唑盐（M1Tr）法检测细胞增殖情况。结果（1）868位点的siRNA抑制效率最高；（2）与正常组相比，siRNA—DDR2转染组DDR2和α-SMA的mRNA表达均显著下降（分别t=6．43，t=7．34，均P〈0．01），DDR2蛋白的表达亦显著降低（t=4．49，P〈0．01），同时HSC的增殖能力也显著降低（t=18．32，P〈0．01）；相反，阴性对照组与正常组相比，DDR2、α-SMA的mRNA表达以及DDR2蛋白和HSC的增殖能力则无明显改变。结论siRNA-DDR2能显著抑制HSC的活化与增殖，进而抑制纤维化发生，具有潜在的抗纤维化作用。
Objective To explore the effects of inhibiting DDR2 expression by siRNA on hepatic stellate cells and evaluate the role of DDR2 gene in hepatic fibrogenesis. Methods （ 1 ） Three pairs of chemically synthesized siRNAs targeting DDP,2 were respectively transfected into HSC-T6 cells for evaluation of silence efficacy, and the most effective siRNA was used. （2） HSC-T6 ceils were divided into three groups, group A served as normal controls, group B served as negative control and group C was RNA interference DDR2 （siRNA-DDR2） expression of HSC. The most effective RNA interference sequences targeting DDR2. gene was chosen to transfect HSC-T6 cells by plasmid transfection. The tendency of DDB2, a-smooth muscle actin （α-SMA） and collagen-I mRNA expression were estimated using RT-PCR, and the protein expression of DDR2 was evaluated by Western blot. Meanwhile, MTT assay was employed to analyze the proliferation of HSC. Results （1） DDR2 siRNA, which began at nt 868, inhibited DDR2 gene expression stronger than the other two siRNAs. （2） After transfection of siRNA-DDR2, the mRNA expression of DDR2 （ P 〈 0.01 ） and α-SMA （ P 〈 0. 01 ） significantly decreased compared with the normal group, and the protein expression of DDR2 also significantly decreased （ P 〈 0. 01 ）. In addition, the proliferation of HSC was also markedly suppressed as compared with the normal group （ P 〈 0. 01 ）. However, compared with the negative control group, none of them was markedly suppressed. Conclusion SiRNA targeting DDR2 significantly suppresses the activation, proliferation of HSC, and thus attenuates hepatic fibrogenesis in vitro.
出处
《中华普通外科杂志》
CSCD
北大核心
2009年第9期748-751,共4页
Chinese Journal of General Surgery
基金
上海市教育委员会课题资助项目（06BZ027）
关键词
RNA干扰
盘状结构域受体2
肝星状细胞
肝硬化
RNA interference
Discoidin domain receptor 2
Hepatic stellate cell
Liver cirrhosis
