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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.
OBJECTIVE: To construct the positive and antisense eukaryotic expression plasmids of KAI1 gene and investigate its effect on KAI1 protein expression in MHCC97-H hepatoma cells with high metastatic potential.Methods: The positive and negative eukaryotic expression plasmids of KAI1 gene were constructed by subcloning technique, The cells were transfected into MHCC97-H hepatoma cell line with high metastatic potential by liposome method, and the expression of KAI1 protein was detected by immunocytochemical SP method. Results: Restriction endonuclease analysis demonstrated that the structures of both recombinants were consistent with the expected structure of the KAI1 sense and antisense gene expression plasmids. Immunocytochemistry SP assay showed that the KAI1 protein in hepatocellular carcinoma cells transfected with sense KAI1 gene was stained darker (IOD20.127 ± 5.099 vs 12.675 ± 1.921, P <0.01) The expression of KAI1 gene in HCC cells was lighter (IOD 8.681 ± 2.472 vs 12.675 ± 1.921, P <0.01) .Conclusion: Positive and antisense eukaryotic expression plasmids for KAI1 gene were successfully constructed.KAI1 gene can upregulate HCC KAI1 protein expression, on the contrary, KAI1 antisense gene is able to down regulate the expression of KAI1 protein in liver cancer cells.