重组质粒p-Egr-p16的构建及在SMMC-7721细胞中的辐射诱导表达

Construction of p-Egr-p16 plasmid and its expression induced by radiation in SMMC-7721 cells

目的 构建重组质粒p Egr p16并探讨在SMMC 772 1细胞中的辐射诱导表达及抗肝癌的作用。方法 用双酶切、粘端连接的方法构建了含有辐射诱导特性的早期反应因子Egr 1和p16的p Egr p16的质粒载体 ,以脂质体介导的方法 ,将重组载体导入人肝癌SMMC 772 1细胞 ,采用RT PCR的方法检测了不同剂量6 0 Coγ射线照射转染后的人肝癌细胞p16基因转录水平的变化和 2Gy照射后不同时间的p16基因转录水平的变化。结果 经研究证实 ,1～ 6Gy照射后p16的转录水平高于对照 ,在 2～ 4Gy照射后达到峰值 ,2Gy照射后不同时间均有所增高 ,在照射后 2 4h增高最明显。结论 2Gy 6 0Coγ射线照射后p16基因转录水平在 2～ 2 4h呈现时间依赖性的增高 ,在 2 4h增高最明显 ,48和 72h逐渐降低 ,但仍高于对照组。提示6 0 Coγ射线可激活早期反应生长因子Egr 1。

Objective To construct a recombinant plasmid p-Egr- p16, investigate the expression of p16 induced by radiation in SMMC-7721 cells and its role in hepatoma suppression. Methods p-Egr-p16 vector that included Egr-1 and p 16 gene was constructed by restriction enzyme digest, ligation and introduced into SMMC-7721 cells by lipofectamine-mediated transfection. Semi-quantitati ve RT -PCR was used to detect the dose-effect and time-course manners of p16 mRNA l evel after irradiation. Results The results showed that p16 mRNA level was higher tha n that of the controls after 1-6 0 Gy irradiation. There was a peak after 2-4 Gy irradiation. p16 mRNA level increased at different times after 2 Gy irradiat ion, and the increase was marked at the 24th hour. Conclusions It is shown that p16 expression levels in SMMC-7721 c ells at 2-48 h after 2 Gy irradiation are higher than those of the sham-irradi at ion group. p16 expression levels show a time-dependant increase from 2 to 24 ho urs, and then a decrease at 48 and 72 h, but it is still higher than the sham-i rradiation group. It is suggested that 60 Co γ-rays could activate Eg r-1 gene promoter and induce the expression of downstream gene p16. ;