表达反义c-myc RNA的重组逆转录病毒载体对人胰腺癌细胞恶性表型的逆转作用

为研究表达反义c－mycRxA的逆转录病毒载体抑制靶基因的表达和翻译，使胰腺癌细胞恶性表型逆转的机理，利用PXTI逆转录病毒载体构建了一个能表达反义c－mycRNA的质粒，经病毒包装细胞PA317包装后，使之成为具有感染力的重组病毒。用该病毒感染人胰腺癌细胞系PC－2细胞，经G418筛选得到稳定的转化细胞系。Northernblot杂交证实包装细胞PA317及转化的PC－2细胞中均有病毒的高表达，体外合成的单链RNA探针杂交结果表明转化PC－2细胞中有高表达的反义c－mycRNA，而内源性c－mycRNA的表达明显受抑；Westernblot分析发现c－myc癌基因产物P62蛋白的表达显著下降。反义c－mycRNA使人胰腺癌细胞生长速率、~3H-胸腺嘧啶掺入、软琼脂集落形成能力以及探鼠致瘤能力等均明显下降。实验结果表明：表达反义c－mycRNA的逆转录病毒载体能有效地抑制靶基因的表达和翻译，使胰腺癌细胞恶性表型部分逆转。

eversal of malignant phenotype of cells of human pancreatic adenocarcinoma cell line by recombinant retroviral vector expressing antisense c-myc

A recombinant retroviral vector expressing antisense c-myc was constructed. The re combinant virus DNA was packaged with packaging cell line PA317 cells by the calcium phosphate co precipitation method. The virus supernatant was used to infect human pancreatic carcinoma cell line PC 2. After selection with G418, resistant colonies were obtained. Stable expression of retrovirus in bothPA317 and PC-2 cells were confirmed by Northern blot hybridization- A down-regulation of endogenousc-myc was found in PC-2 cells infected with antisense c-myc construct. It was dem0nstrated that the an tisense c-myc did inhibit the cell growth rate and 3 H-TdR incorporation rate 0f PC-2 cells. The abilityof colony formation in soft agar and tumorogenicity in nude mice of PC-2 cells were significantly sup pressed by the antisense c-myc. The results implicate that recombinant retroviral vector containing anti sense c-myc could inhibit target gene expression and partly reverse malignant phenotype of pancreaticadenocarcinoma cells.