论文部分内容阅读
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.
OBJECTIVE: To construct eukaryotic expression plasmids targeting short hairpin RNA (shRNA) targeting signal adapter Crk and study its effect on the function of hepatic stellate cell line LX-2.Methods: According to the sequence of Crk mRNA ShRNA oligonucleotide fragments were designed and synthesized, annealed to form double-stranded chains and ligated into plko vector.The Crk lentivirus was packaged to infect human hepatic stellate cell line LX-2, and its function on proliferation, migration and activation of LX-2 cells was studied .Results: The results of quantitative PCR and Western blot showed that the expression of Crk-specific shRNA eukaryotic expression vector was successfully constructed, and the expression of Crk in shCrk group was significantly lower than that in control group.After interference with Crk gene, the activation and migration of LX-2 cells Decreased, while cell proliferation was not affected.Conclusion: The shRNA eukaryotic expression vector of Crk gene was successfully constructed in LX-2, and downregulation of Crk gene could inhibit the activation and migration of LX-2 cells.