67kD层粘连蛋白受体的克隆、表达及其对肝癌细胞侵袭能力的影响

Cloning, Expression of 67 kD Laminin Receptor and Its Effect on Invasiveness of Human Hepatocellular Carcinoma Cells

探讨细胞膜表面 6 7kD层粘连蛋白受体 (6 7kDlamininreceptor ,6 7LR)在肝癌细胞侵袭转移中的作用 ,从肝癌细胞中提取RNA ,通过RT PCR扩增 6 7LR的前体——— 37kD层粘连蛋白受体前体(37kDlamininreceptorprecursor,37LRP)基因并定向克隆到真核表达载体pcDNA3.1 myc His(- )A ,采用脂质体将重组质粒转染到HepG2肝癌细胞中 ,通过G4 1 8筛选和RT PCR、流式细胞术鉴定 ,获得了细胞膜表面 6 7LR高表达 (阳性率为 6 9.2 % )和低表达 (阳性率为 1 1 .7% )的细胞克隆 ,采用体外细胞侵袭实验测定不同细胞的侵袭能力 ,发现膜表面 6 7LR高表达的细胞侵袭能力明显高于低表达及不表达细胞 ,说明 6

In order to explore the effect of 67 kD laminin receptor on invasiveness of human hepatocellular carcinoma cells, 37 kD laminin receptor precursor(37LRP)gene was amplified by RT PCR using the total RNA extracted from HCC cells. The recombinant plasmid was constructed with pcDNA3.1/myc His( ) A using Bam H Ⅰ, Nhe I and was transformed into E. coli Top10. The positive clones were identified by restriction enzymes and DNA sequence analysis. The correct recombinant plasmid was transfected into HepG2 HCC cells and was screened by G418. The expression of 67 kD laminin receptor was detected by RT PCR and flow cytometric analysis. A high expression of membrane 67LR cell clone which positive rate was 69.2%(clone number: LR4) and a low one which positive rate was only 11.7%(clone number: LR6) were obtained and used to assay the invasion potential in a transwell culture chamber besides HepG2 with or without being transfected by pcDNA3.1/myc His( ) A. The LR4 cells showed the highest invasion potential ( P <0 001), while the LR6 cells and HepG2 with or without being transfected by pcDNA3.1/myc His( )A had no significant difference( P >0.05). The results indicated 67LR had an important role in the invasion and metastasis of hepatocellular carcinoma cells.