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目的:构建CD44新剪接变异体siRNA质粒表达载体,建立CD44新变异体抑制表达的鼻咽癌细胞株。方法:合成CD44新变异体特异性干扰DNA片段,干扰DNA片段亚克隆于带绿色荧光蛋白的pGenesil-1.3质粒表达载体中,双酶切和测序鉴定重组表达质粒载体;采用脂质体将重组表达质粒载体转染入鼻咽癌5-8F细胞系,进行G418筛选;Western blot分析CD44表达。结果:重组CD44干扰DNA片段质粒表达载体的碱基序列和插入方向正确;细胞转染效率达70%;G418筛选获得GFP表达的单克隆鼻咽癌5-8F细胞株;Western blot分析表明CD44表达受抑制。结论:建立了CD44新变异体抑制表达的鼻咽癌细胞株,为CD44新变异体在鼻咽癌中的生物学功能提供了基础。
OBJECTIVE: To construct siRNA plasmid expression vector of CD44 new splice variants and to establish nasopharyngeal carcinoma cell lines with novel expression of CD44. Methods: The new variant of CD44 was synthesized to interfere with the DNA fragment. The DNA fragment was subcloned into pGenesil-1.3 plasmid with green fluorescent protein. The recombinant plasmid was identified by double enzyme digestion and sequencing. Recombinant plasmid The plasmid vector was transfected into nasopharyngeal carcinoma cell line 5-8F and then screened by G418. CD44 expression was analyzed by Western blot. Results: The sequence and insertion direction of the recombinant plasmids of CD44 interference DNA fragment were correct. The efficiency of transfection was 70%. The expression of CD44 was detected by G418 in 5-8F cell line of monoclonal nasopharyngeal carcinoma Suppressed. CONCLUSION: The nasopharyngeal carcinoma cell line with CD44 expression is inhibited, and the new CD44 variant provides the basis for the biological function of NPC.