RNA干扰Ku70基因对宫颈癌Hela细胞放射敏感性影响的实验研究

Effect of Ku70 gene interfering RNA on radiation sensitivity of cervical cancer Hela cells

目的观察ku70基因短干扰RNA(siRNA)对宫颈癌Hela细胞放射敏感性的影响。方法采用集落形成技术测定不同剂量射线照射对Hela细胞存活率的影响,观察Hela细胞对射线照射的敏感性。采用时定量PCR技术(quantitiv real time PCR,QRT-PCR)检测Ku70 mRNA水平表达。采用Western Blot免疫印记技术检测Ku70蛋白表达。采用RNA干扰(RNAi)技术抑制Hela细胞中Ku70蛋白的表达,观察Ku70蛋白表达下调对Hela细胞放射敏感性的影响。采用集落形成技术测定4.0 Gy射线照射对Hela细胞存活率的影响。进而分析Hela细胞对射线的放射敏感性。结果宫颈癌Hela细胞对1.0-4.0 Gy射线照射不够敏感,有相当高的辐射抗性。量效实验证实,1.0-6.0 Gy射线照射后,HeLa细胞中Ku70 mRNA及Ku70蛋白表达均明显增高。时效实验证实,4.0 Gy射线照射后8 h-72 h,HeLa细胞中Ku70 mRNA及Ku70蛋白表达均明显增高。本实验成功的构建了Ku70基因RNA干扰载体pSilencer-4.1-Ku70,稳定转染HeLa细胞后,有效抑制Ku70蛋白的表达。稳定转染pSilencer-4.1-Ku70干扰质粒并抑制Ku70蛋白表达后,HeLa细胞对射线照射的放射敏感性显著增高(P<0.001)。结论 Ku70 siRNA有效下调Ku70 mRNA水平,抑制Ku70蛋白的表达,可明显提高Hela细胞对辐射的敏感程度。本研究结果还提示,Ku70基因可作为衡量宫颈腺癌辐射敏感性指标,为临床合理筛选患者进行放疗、预测放疗疗效提供一条适用途径。

Objective To observe the ku70 gene short interfering RNA（siRNA） on cervical cancer Hela cells,radiation sensitivity of.Methods Determination of colony-forming technology,different doses of-ray irradiation effect on the Hela cell viability was observed Hela cells to-ray radiation sensitivity.When using quantitative-PCR（quantitiv real time PCR,QRT-PCR） detection of expression of Ku70 mRNA level.Western-blot technique using Western Blot Detection of Ku70 protein expression.Using RNA interference（RNAi） techniques to suppress the Hela cells,Ku70 protein expression was observed decreased expression of Ku70 protein in Hela cells on radiation sensitivity.Colony formation was measured by using 4.0 Gy-ray irradiation effect on the survival rate of Hela cells.Further analysis of Hela cells,the radiosensitivity of -rays.Results The cervical cancer Hela cells to 1.0～4.0 Gy-rays is not sensitive enough,there is a very high radiation resistance.Dose-response experiments confirmed that,1.0～6.0 Gy-ray irradiation,HeLa cells and the Ku70 protein expression of Ku70 mRNA were significantly increased.Aging experiments confirmed that,4.0Gy-ray irradiation 8 h～72 h,HeLa cells and the Ku70 protein expression of Ku70 mRNA were significantly increased.In this study,the successful construction of the Ku70 gene RNA interference vector pSilencer-4.1-Ku70,stably transfected HeLa cells,effectively inhibit the expression of Ku70 protein.Stable transfection of pSilencer-4.1-Ku70 interfere with and inhibit the Ku70 protein expression plasmid,after,HeLa cells rays of radiation sensitivity was significantly higher（P〈0.001）.Conclusion Ku70 siRNA effectively reduced the level of Ku70 mRNA,inhibit the expression of Ku70 protein can significantly enhance the Hela cells,the degree of sensitivity to radiation.The results of this study also suggests,Ku70 gene can be used as indicators to measure radiation sensitivity of cervical cancer,screening patients for clinical radiotherapy,forecasting to provide a suitable means of radiotherapy.