目的 构建融合蛋白SMP30-IP10真核表达质粒,并初步探究其对肝癌细胞株SK-hep1迁移、侵袭及增殖的影响.方法 将肝癌细胞株SK-hep1分为两组,实验组转染pIRES-SMP30-IP10质粒,对照组转染空质粒pIRES;通过重叠PCR方法构建pIRES-SMP30-IP10真核表达质粒;采用脂质体转染法转染肝癌细胞SK-hep1;Western blot-ting法检测蛋白表达;Transwell法检测细胞的迁移和侵袭能力;CCK8法检测细胞增殖能力.结果 通过PCR、双酶切及测序鉴定证实成功构建了真核表达质粒pIRES-SMP30-IP10,并在肝癌细胞株SK-hep1中正确表达.与对照组相比,实验组细胞迁移、侵袭能力下降(P均<0.05),但细胞形态及增殖能力均差异无统计学意义(P均>0.05).结论 成功构建了融合蛋白SMP30-IP10真核表达质粒,融合蛋白SMP30-IP10可抑制肝癌细胞的迁移、侵袭能力,但对细胞的增殖能力影响不明显.%Objective To construct the eukaryotic expression plasmid of fusion protein SMP30-IP10,and to investi-gate its effects on migration,invasion,and proliferation of hepatoma cell line SK-hep1. Methods The SK-hep1 cells were divided into two groups:the experimental group which was transfected with pIRES-SMP30-IP10 plasmid,and the con-trol group which was transfected with empty pIRES. The eukaryotic expression plasmid pIRES-SMP30-IP10 was constructed by overlapping PCR,and then we transfected the plasimd into hepatoma cell line SK-hep1 by liposome transfection;the protein expression of cells was detected by Western blotting;the migration and invasion of cells were detected by Transwell assay;the proliferation of cells was detected by CCK8. Results The eukaryotic expression plasmid pIRES-SMP30-IP10 was successfully constructed and approved by PCR,double restriction enzyme digestion and DNA sequencing,and was ex-pressed in hepatocellular carcinoma cell line SK-hep1. Compared with the control group,the migration and invasion were inhibited to some extent in the experimental group (both P < 0. 05),but the morphology and proliferation had no significant change (both P > 0. 05). Conclusion The fusion protein SMP30-IP10 eukaryotic expression plasmid is successfully con-structed,and it can inhibit the migration and invasion abilitiesof hepatoma cells,but has no effect on the cell proliferation.
展开▼
