摘要
目的:构建KAI1基因正、反义真核表达质粒,了解其对高转移潜能的MHCC97—H肝癌细胞KAI1蛋白表达的影响.方法:利用亚克隆技术构建KAI1基因正、反义真核表达质粒,并用脂质体法将其分别转入高转移潜能的MHCC97-H肝癌细胞系,通过免疫细胞化学SP法检测KAI1蛋白表达情况。结果:限制性内切酶分析证明两个重组子的结构均与KAI1正、反义基因表达质粒的预期结构一致。免疫细胞化学SP法检测显示,转入正义KAI1基因后的肝癌细胞KAI1蛋白染色加深,(细胞积分光密度 integra oculus dehter,IOD20.127 ± 5.099 vs 12.675±1.921,P<0.01);而转入反义KAI1基因的肝癌细胞则KAI1蛋白染色变浅,(IOD 8.681±2.472 vs 12.675 ± 1.921,P<0.01).结论:成功构建了KAIl基因正、反义真核表达质粒.KAI1正义基因能上调肝癌细胞KAI1蛋白的表达,相反,KAI1反义基因则能下调肝癌细胞KAI1蛋白的表达.
AIM: To construct sense and antisense KAI1 expression plasmids and to explore their effects on KAI1 protein expression in MHCC97-H hepatocellular carcinoma cells with high metastatic potential. METHODS: Sense and antisense KAI1 expression plasmids were constructed using subclone technique and transfected into MHCC97-H cells by DOTAP liposome system. KAI1 protein expression in transfected MHCC97-H cells was analyzed with immunocytochemical SP method. RESULTS: Analysis of restriction endonuclease indicated the structure of two recombinants was consistent with the expected results of sense and antisense KAI1 expression plasmids. Compared with MHCC97-H cells, KAI1 protein staining in the MHCC97-H-S cells transfected with sense KAI1 expression plasmid was obviously enhanced(Integra Oculus Dehter ,IOD 20.127±5.099 vs 12.675±1.921,P<0.05). However, KAI1 expression in the MHCC97-H-AS cells trans- fected with KAI1 antisense gene was obviously weakened (IOD 8.681±2.472 vs 12.675±1.921, P<0.05). CONCLUSION: We successfully constructed sense and antisense KAI1 expression plasmids. Sense KAI1 gene can upregulate the expression of KAI1 protein in MHCC97-H cells. On the other hand, antisense KAI1 gene can downregulate the expression of KAI1 protein in MHCC97-H cells.
出处
《世界华人消化杂志》
CAS
2003年第9期1341-1344,共4页
World Chinese Journal of Digestology
基金
国家自然科学基金
No.30070348
