反义核酸抑制胃癌细胞Bcl-2表达

Expression of Bcl-2 in Gastric Cancer Cells Transducted with Antisense Bcl-2

本文采用分子克隆技术成功地构建了反义核酸表达载体ｐＤＯＲ－Ｂｃｌ－２ ｃＤＮＡ。以奚质体介导法将重组反义核酸表达载体转染受体细胞ＳＧＣ７９０１，并Ｇ４１８筛选，从２×１０＾５细胞中筛选出大约１５０个抗性克隆，转导率大于１‰，随机挑选２个克隆继续筛选与扩增培养，获得了１株稳定的抗性细胞，从而建立了Ｂｃｌ－２表达阻断的胃癌细胞株，我们命名为７９０１ａｎＢｃｌ－２ｃｅｌｌｓ。通过Ｓｏｕｔｈｅｒｎ印迹法、

By molecular cloning technique, the expressing plasmids pDOR-SV40-Bcl-2 cDNA were successfully constructed. The reconstructed plasmids with lipofectamine were transduced into gastric cancer cell line SGC7901 and then the positive clones which contained the reconstructed plasmids were choosed by G418. Circa 150 positive clones were choosed from 2 x 10_5 gene transduced cells, which suggested that the transducing efficiency was more than 1%o; 2 positive clones were expanded and passage, then 1 drug-resistance cell strain (SGC7901 anBcl-2 cells) was obtained. The bloting results suggested, Bcl-2 cDNA were expressed in both gene transduced and not transduced cell strains, but the expressing level of mRNA and protein in gene transduced cell strain was very low than that in gene not transduced cell strain were positive by the means of Southern blot, Northern blot and Western blot. This results showed that normally in gastric cancer cells the expressing level of Bcl-2 gene was very high, and the cDNA fragment of antisense Bcl-2 were successfully transduced into gastric cancer cells, and in gene transduced cell strains the expressing of Bcl-2 gene was effi-cientlv blocked.