smac基因的克隆及过表达对宫颈癌HeLa细胞γ射线敏感性的影响

Cloning of smac gene and its overexpression effects on radiosensitivity of HeLa cells to γ-rays

目的克隆smac(thesecondmitochondria-derivedactivatorofcaspases,smac)基因,构建真核表达载体pcDNA3·1/smac,并转染宫颈癌HeLa细胞,探讨smac基因过表达对宫颈癌HeLa细胞γ射线敏感性的影响。方法用RT-PCR的方法从HeLa细胞总RNA中克隆smac基因,连入pcDNA3·1载体并测序。将测序正确的pcDNA3·1/smac载体转染HeLa细胞系,利用RT-PCR法及Westernblot鉴定HeLa细胞内smac基因的表达。γ射线照射后48h利用MTT法检测各组细胞生长抑制率。结果测序结果表明成功的从HeLa细胞总RNA中扩增了全长smac基因。RT-PCR及Westernblot结果表明,转染pcDNA3·1/smac的HeLa/smac细胞中,smac基因表达量明显高于未转染的HeLa细胞及转染空载体pcDNA3·1的HeLa/pcDNA3·1细胞的表达量。不同剂量的γ射线照射48h后,MTT结果表明,γ射线对转染pcDNA3·1/smac的HeLa/smac细胞的生长抑制率(38·85%)显著高于空白组HeLa细胞(17·64%)及转染空载体pcDNA3·1的HeLa/pcDNA3·1细胞(20·32%)。结论成功地克隆了smac基因,在宫颈癌HeLa细胞中过表达外源smac基因能够提高其对γ射线的敏感性,有望开辟提高宫颈癌放疗敏感性的新途径。

Objective To clone smac gene and construct eukaryocytic expression vector pcDNA3.1/ smac. The smac gene was transfected into HeLa cells to explore the effects of over-expression of extrinsic smac gene on radiosensitivity to γ-rays of HeLa cells. Methods The full-length smac gene was amplified from total RNA of HeLa cells by RTPCR. The RTPCR product was ligated with the vector pcDNA3.1 and sequenced. The correct pcDNA3.1/smac was transfected into HeLa cells. The expression of smac gene was tested by RTPCR and Western blot. The cellular growth inhibition rates were evaluated by MTT 48 hours after irradiation with different doses of γ-rays. Results Recombinant eukaryocytic expression vector pcDNA3.1/smac was successfully constructed. RTPCR and Western blot results indicated that the expression of smac gene of HeLa/smac cells was significantly enhanced compared with the expression of smac gene of HeLa/pcDNA3.1 and HeLa cells. 48 hours after different doses of γ-ray irradiation was significantly higher in pcDNA3.1/smac transfected HeLa/smac cells than those of non-transfected HeLa ceils or pcDNA3.1 transfected HeLa/pcDNA3.1 ceils, inhabitation rates were 38.85% ,17.64%and 20.32% ,respectively. Conclusions smac gene was successfully cloned. Extrinsic smac gene over-expression could significantly enhance radiosensitivity to γ-ray of HeLa cells, which would herald a new approach to improve radiosensitivity of cervical cancer.