摘要
目的转染pCMV-KAI1重组质粒人人胰腺癌细胞株MiaPacaⅡ和Panc1,观察KAI1基因的表达,为进行KAI1基因在胰腺癌中抗转移作用的有关研究提供实验用靶细胞,并为今后利用KAI1基因治疗胰腺癌提供实验和理论依据。方法采用Westernblot方法从7种胰腺癌细胞中筛选出无KAI1表达的MiaPacaⅡ和Panc1细胞株,通过脂质体转染法将pCMV-KAI1重组质粒转染到MiaPacaⅡ和Panc1中,经G418筛选4周,获得单克隆细胞系,应用Westernblot、免疫荧光及免疫细胞化学方法检测转染细胞系KAI1蛋白的表达。结果获得了稳定表达KAI1基因的MiaPacaⅡ和Panc1胰腺癌细胞克隆;经Westernblot分析显示转染后的胰腺癌细胞株MiaPacaⅡ与Panc1有明确的分子质量为29100KAI1蛋白表达,而未转染细胞无KAI1蛋白表达;免疫荧光显示,转染的胰腺癌细胞质中可见明显的荧光,而无转染的母细胞无荧光;免疫细胞化学检测显示,无转染的细胞呈阴性反应,转染的细胞呈中到强度的阳性反应。结论pCMV-KAI1重组质粒经转染入人胰腺癌细胞株MiaPacaⅡ和Panc1后可表达KAI1蛋白,从而建立了KAI1蛋白表达的MiaPacaⅡ和Panc1细胞系。
Objective To transfect recombinant plasmid pCMV-KAI1 to Panc 1 and MiaPaca Ⅱpancreatic cancer cells for studying the expression of KAI1 gene. The transfected cells are expected to be used as the target cell in the study of anti-metastatic role of KAI1 gene in human pancreatic cancer, thus providing experimental and theoretic foundation for gene therapy of human pancreatic cancer with KAI1 gene. Methods The expression of KAI1 protein was negative in Panc 1 and MiaPaca Ⅱ of seven kinds of pancreatic cancer cells with Western blot, so Panc 1 and MiaPaca Ⅱ were transfected with pCMV-KAI1 by lipofectamine, and then selected by G418; the expression of KAI1 in the monoclone cells obtained was detected by Western blot, immunofluorescence staining and immunocytochemistry. Results Stably transfected cells were obtained. Western blot showed that there was expression of KAI1 protein in 29100 D inside the transfected Panc 1 and MiaPaca Ⅱpancreatic cancer cells; KAI1 protein was found by immunofluorescence staining and immunocytochemistry in the transfected pancreatic cancer cells. Conclusions KAI1 can be expressed effectively in Panc 1 and MiaPaca Ⅱ pancreatic cancer cells transfected by recombinant plasmid pCMV-KAI1. The metastatic mechanism of transfected pancreatic cancer cells can be explained to be concerned with that of patients with pancreatic cancer.
出处
《胰腺病学》
2004年第3期141-144,共4页
Chinese JOurnal of Pancreatology
基金
国家自然科学基金资助项目(No.39970344)
