外源p21~(WAF1)转染对人成纤维细胞凋亡的影响

构建了有义和反义ｐ２１ＷＡＦ１ 逆转录病毒表达载体， 分别经脂质体包裹后转染人胚肺二倍体成纤维细胞（２ＢＳ）。Ｓｏｕｔｈｅｒｎ 印迹杂交证实转染细胞中外源ｐ２１ ＷＡＦ１ｃＤＮＡ 已整合入基因组中。与空载体转染细胞相比， 有义转染细胞的ｐ２１ＷＡＦ１ ｍ ＲＮＡ 表达上升； 细胞增殖速度明显减慢； 对丁酸钠诱导凋亡的敏感性降低， 表现在细胞存活率升高， 核ＤＮＡ 梯状断裂片段出现的时间滞后， 断裂片段浓度下降， 流式细胞计检测的凋亡峰面积缩小。而反义转染细胞的ｐ２１ＷＡＦ１ ｍ ＲＮＡ表达下降； 细胞增殖速度较快； 对丁酸钠诱导凋亡的敏感性上升， 有关表现与有义转染细胞相反。说明２ＢＳ细胞内ｐ２１ＷＡＦ１ 的表达量与其被丁酸钠诱导凋亡的能力呈负相关。

Exogenous p21~(WAF1) Transfection Affects Apoptosis in Human Fibroblasts

pDOR p21sense and pDOR p 21 antisense retroviral expression vectors were constructed and successfully transfected by Lipofectin into normal human diploid fibroblasts(2BS line), resulting in 2BS p 21s and 2BS p 21a cell lines, respectively. Southern blot analysis verified that the exogenous p 21 WAF1 cDNA were integrated into genomic DNA. Compared with the cells transfected with pDOR neo empty vector, p 21 WAF1 mRNA expression increased in 2BS p 21s cells, which were less sensitive to apoptosis induced by NaBu, and showed higher cell viability, delayed appearance of DNA ladder, and less area of apoptosis peak. On the other hand, p 21 WAF1 mRNA expression decreased in 2BS p 21a cells, which were more sensitive to apoptosis induced by NaBu. These results indicated that the expression amount of p21 WAF1 in 2BS cells was negatively related with its susceptibility to apoptosis induced by NaBu.