【作者】 李翀；

【导师】 曹建平；

【作者基本信息】 苏州大学 ， 放射医学， 2007， 硕士

【摘要】 目的利用化学合成的siRNA抑制Hela细胞ATM基因的表达,建立ATM基因沉默的细胞模型,即Hela^ATM-细胞;利用常规染色体畸变分析技术和胞质分裂阻滞微核法,通过与Hela细胞比较,研究Hela^ATM-细胞的放射敏感性。本课题为研究ATM基因沉默的肿瘤细胞的生物学特性提供了细胞模型,探讨了ATM基因对肿瘤细胞放射敏感性的影响,为研究如何提高肿瘤放射敏感性提供实验思路和实验依据。方法（1）设计合成人ATM基因的4对siRNA、siRNA阴性、siRNA阳性对照及FAM标记阴性对照siRNA各1对,脂质体法介导转染Hela细胞;倒置荧光显微镜下观察转染结果; RT-PCR方法检测转染siRNA 24h后Hela细胞中ATM基因的表达水平;筛选干扰效果明显的siRNA,转染Hela细胞,分别检测转染后24h、48h、72h和96h的Hela细胞中ATM基因的表达水平,观察干扰效果的持续时间;建立ATM基因沉默的Hela^ATM-细胞模型。（2）采用常规染色体畸变分析法,在Hela^ATM-细胞和Hela细胞经60Coγ射线0、1、2、3、4和5Gy照射后,观察比较两种细胞的染色体畸变率的差异;分别建立Hela^ATM-细胞和Hela细胞染色体畸变的剂量效应曲线。（3）采用胞质分裂阻滞微核法,在Hela^ATM-细胞和Hela细胞经60Coγ射线0、1、2、3、4和5Gy照射后,观察比较两种细胞的微核率和微核细胞率的差异;分别建立Hela^ATM-细胞和Hela细胞微核率和微核细胞率的剂量效应曲线。结果（1）荧光显微镜下观察到FAM标记的siRNA阴性对照定位在Hela细胞中,表明siRNA成功转染进Hela细胞。（2）RT-PCR结果显示,转染siRNA 24h后,转染序列为1057-1075nt siRNA的Hela1057细胞中ATM基因的表达受到明显抑制（P <0.05）,建立ATM基因沉默的Hela1057细胞模型。（3）RT-PCR结果显示,与Hela细胞相比,Hela1057细胞的siRNA干扰效果持续至转染后96h（P<0.05）;Hela1057细胞中ATM基因的表达在转染siRNA后24h与48h之间以及72h与96h之间无显著性差异（P>0.05）;24h、48h的ATM基因表达水平明显低于72h、96h的表达水平（P <0.05）。（4）在0、1、2、3、4和5Gy照射剂量内,Hela^ATM-细胞和Hela细胞以双着丝粒体为主要染色体畸变类型;各照射剂量点,Hela^ATM-细胞染色体畸变率明显高于Hela细胞（P<0.01）;Hela^ATM-细胞和Hela细胞染色体畸变率与剂量呈正相关,均可拟合成直线方程Y=a+bD模式,且Hela^ATM-细胞剂量效应直线回归方程斜率明显大于Hela细胞（P<0.05）。（5）在1、2、3、4和5Gy剂量照射下,Hela^ATM-细胞微核率及微核细胞率均明显高于Hela细胞（P<0.01）,并且两者微核率及微核细胞率与剂量的关系拟合成剂量效应方程为y=a+bD+cD2模式。结论（1）化学合成的siRNA转染Hela细胞,可以明显抑制ATM基因的表达,并建立了ATM基因沉默的Hela ATM-细胞模型;（2）ATM基因沉默的Hela^ATM-细胞的染色体畸变率明显高于Hela细胞;（3）ATM基因沉默的Hela^ATM-细胞的微核率和微核细胞率也明显高于Hela细胞。更多还原

【Abstract】 Objective To construct the Hela cell model with ATM gene silenced(Hela^ATM-cell) by transfecting chemically synthesized siRNA. To study the radiosensitivity of Hela cells with ATM gene silenced by using conventional chromosome aberration analysis method and cytokinesis-block micronucleus method. This research provided a cell model for further research on the bionomics of tumor cells with ATM silenced,and present a new idea and experimental data for the research on the radiosensitivity of tumor cells.Methods （1） siRNA was designed and synthesized ,including 4 pairs of siRNA targeting ATM gene,the negative and positive control siRNA and one pair of FAM-marked negative control siRNA. Transfected these siRNA into Hela cells by using liposome. Transfection effect was observed under Fluorescent Microscope. Using RT-PCR to detect ATM gene expression of Hela cells 24h after siRNA transfected. Transfected the more effective siRNA into Hela cells and detected ATM gene expression respectively after 24h、48h、72h and 96h.Constructed the Hela^ATM-cell with ATM gene silenced . （2） Using conventional chromosome aberration analysis method,chromosome aberration frequencies （CAF） of Hela^ATM-cells and Hela cells with 0、1、2、3、4 and 5 Gy exposures to 60Coγ-ray were observed,compared with Hela cells. （3） Using cytokinesis-block micronucleus method,the micronucleus frequencies （MNF） and micronucleus cell frequencies （MNCF） of Hela^ATM-cells and Hela cells with 0、1、2、3、4 and 5 Gy exposures to 60Coγ-ray were observed,compared with Hela cells.Results （1） It showed that FAM-marked negative control siRNA was successfully transfected into Hela cells under Fluorescent Microscope. （2） RT-PCR showed that ATM gene expression was depressed significantly in Hela1057cells which had been transfected by 1057-1075nt siRNA 24h before （P<0.05）. Hela^ATM-cell with ATM gene silenced was constructed .（3） RT-PCR showed the 1057-1075nt siRNA worked untill 96 hours after transfected （P<0.05）. ATM gene expression was decreased significantly at 24h and 48h after transfected than 96h （P<0.05）,no significant difference between 24h and 48h,72h and 96h after transfected （P>0.05）;ATM gene expression was decreased significantly at 24h and 48h than 72h and 96h after transfection （P<0.05）. （4） After exposed to 0、1、2、3、4 and 5 Gy ⁶⁰Coγ-ray,the main pattern of chromosome aberration was dic and the radiation-induced level of CAF was significantly higher in Hela^ATM- cells than in Hela cells at every expose dose point （P<0.01） . In the two cells,CAF had a positive correlation with dose,and their linear regression equations were Y=a+bD. The slope of CAF linear regression equations of Hela^ATM- cells was larger than that of Hela cells （P<0.05）. （5） After exposed to 0、1、2、3、4 and 5Gy 60Coγ-ray, the radiation-induced levels of MNF and MNCF were significantly higher in the Hela^ATM-cells compared with the Hela cells （P<0.01）. In the two cells, the MNF and MNCF had a correlation with dose,and their linear regression equations were Y=a+bD+cD².Conclusion （1） Hela^ATM-cell with ATM gene silenced was constructed by transfecting chemically synthesized siRNA. （2） The CAF of Hela^ATM- cells was significantly higher than Hela cells by using conventional chromosome aberration analysis method. （3） The MNF and MNCF of Hela^ATM- cells were significantly higher than Hela cells by using Cytokinesis-Block micronucleus method.更多还原