Crk基因shRNA质粒的构建及其在肝纤维化中的作用

Construction of an shRNA expression vector targeting the Crk gene: Potential role of Crk in liver fibrosis

目的:构建靶向信号接头蛋白Crk的短发夹RNA(short hairpin RNA,shRNA)的真核表达载体质粒,研究其对肝星状细胞株LX-2功能的影响.方法:根据Crk mRNA序列进行设计并合成shRNA寡核苷酸片段,退火形成双链并连接入plko载体.包装Crk慢病毒,感染人肝星状细胞株LX-2.研究其对LX-2细胞增殖、迁移和活化功能的影响.结果:定量PCR和Western blot检测结果提示成功构建了针对Crk特异性shRNA真核表达质粒,shCrk组的Crk表达明显低于对照组.干扰Crk基因后,LX-2细胞的活化、迁移能力减弱;而细胞增殖没有受到影响.结论:LX-2中成功构建Crk基因的shRNA真核表达载体;下调Crk基因后能够抑制LX-2细胞的活化和迁移.

AIM： To construct a short-hairpin RNA（shRNA）eukaryotic expression vector targeting the v-Crkavian sarcoma virus CT10 oncogene homologgene（Crk） and to study the potential role of Crkin liver fibrosis. METHODS： The shRNA oligonucleotide frag-ments were designed and synthesized basedon the sequence of Crk mRNA. Double strands were then formed after annealing and inserted into the plko vector. Recombinant lentiviral vec-tor was transfected into 293 T cells to package lentivirus. LX-2 cells were then infected with the recombinant lentivirus and the function of Crk was studied after infection. RESULTS： RT-PCR and Western bolt analyses indicated that after successful infection, both mRNA and protein expression was dramatically down-regulated, compared with the control group. Knockdown of Crk decreased the expres-sion of collagen type 1（Col1）, α-smooth muscle actin（α-SMA） and the capacity of cell migration, but had no effect on cell proliferation. CONCLUSION： We have successfully con-structed an shRNA eukaryotic expression vector targeting the Crk gene. Knockdown of Crk can inhibit liver fibrosis possibly by suppressing the activation and migration of LX-2 cells.