【作者】 王巍；

【导师】 祝淑钗；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2011， 硕士

【摘要】 目的:检测体外培养食管癌细胞中H2AX、STAT1照射前后mRNA及蛋白水平的变化,探讨H2AX、STAT1表达与放射线的时间剂量效应关系,为放射增敏的基因疗法提供理论依据。方法:(1)在人食管癌细胞株TE1、TE13和Eca109细胞中,采用逆转录-聚合酶链反应(RT-PCR)检测H2AX和STAT1 mRNA表达,免疫组织化学、western blotting和流式细胞技术(FCM)检测H2AX和STAT1蛋白表达;(2)采用克隆形成实验检测TE13和Eca109食管癌细胞的放射敏感性;(3)食管癌细胞株TE13、Eca109在4Gy照射后0-24h及0-10Gy照射后0.5h,应用western blotting检测γ-H2AX和pSTAT1蛋白表达的变化;(4)应用激光共聚焦显微镜检测Eca109食管癌细胞照射后γ-H2AX和pSTAT1斑点形成情况及不同剂量-时间照射后γ-H2AX斑点的动态变化。结果:(1) TE1、TE13和Eca109这3株食管癌细胞中,H2AX在mRNA水平和蛋白水平上均有表达,3种食管癌细胞之间H2AX的表达未见明显差异(P>0.05)。STAT1在TE13和Eca109食管癌细胞中采用免疫组织化学未检测到,但是应用逆转录-聚合酶链反应(RT-PCR)、western blotting和流式细胞技术(FCM)检测,显示在3种细胞中均有表达,且之间表达无明显差异(P>0.05);(2)克隆形成实验发现TE13和Eca109细胞接种效率分别为54.62 %和67.68 %,D0值分别为2.35Gy和3.04Gy,SF2值分别为0.752和0.796,提示TE13细胞放射敏感性高于Eca109细胞;(3) Western blotting结果显示,食管癌细胞TE13、Eca109中pSTAT1蛋白在0Gy -10Gy照射后0.5h及4Gy照射后0-24h,表达均没有明显变化(P>0.05);(4)Western blotting结果显示,食管癌细胞TE13、Eca109在0Gy -10Gy照射后0.5h,随照射剂量增加,γ-H2AX表达量逐渐增加,各剂量组与对照组比较有显著性统计学差异(p<0.05),γ-H2AX蛋白表达量与照射剂量存在正相关关系,2种食管癌细胞TE13、Eca109的确定系数分别为:0.923、0.957;在4Gy照射后0-24h条件下结果表明照射后0.5h,γ-H2AX表达达到峰值,之后随时间延长γ-H2AX表达逐渐下降,至24h基本达到未照射水平,各剂量组与对照组比较有显著性统计学差异(p<0.05);(5)应用激光共聚焦显微镜检测显示,食管癌细胞Eca109在4Gy照射后5min即有γ-H2AX斑点形成,照射后0.5h达最大值,之后斑点数量逐渐减少,至24h斑点基本消失。在0Gy-10Gy照射后0.5h,随照射剂量增加,γ-H2AX斑点数量逐渐增多;而STAT1在照射后未见核内斑点的形成,顺铂药物处理后可观察到STAT1核内斑点的形成,但电离辐射+顺铂共同处理后核内斑点数量无明显变化。结论:1、H2AX和STAT1在3种食管癌细胞TE1、TE13及Eca109 mRNA水平和蛋白水平上均有表达;2、Eca109细胞放射抗拒性高于TE13细胞;3、放射线作用后对STAT1蛋白的磷酸化及核内斑点未见影响,经顺铂药物处理后可观察到STAT1核内斑点的形成,但是与放射线联合后斑点数量未见变化;4、γ-H2AX蛋白表达及H2AX核内斑点与放射线存在时间剂量效应关系。更多还原

【Abstract】 Objective: To observe the expression of STAT1 and H2AX mRNA and protein after irradiated in different type of esophageal cancer cells in vitro, in order to test the dose-effects relationship between ionizing radiation intensity and nuclear foci of the STAT1 and H2AX, for providing a base for changing the radio sensitivity of esophageal caicinoma Toad toxinells by gene therapy.Methods: (1) In three human esophageal carcinoma cell lines of TE1, TE13 and Eca109, expression of STAT1 and H2AX mRNA were detected with reverse transcriptase-polymerase chain reaction (RT-PCR), expression of STAT1 and H2AX protein were measured with immunohistochemistry assay, western blotting and indirect immuno-fluorescence assay respectively. (2)Radiosensitivity was analysized with clonegentic assay in TE13 and Eca109 esophageal caicinoma cells in vitro. (3) Expression of STAT1 and H2AX protein were detected with western blotting after irradiated in TE13 and Eca109 esophageal caicinoma cells in vitro. (4) The location and quantity with nuclear foci of STAT1 and H2AX were detected with Zeiss fluorescence microscope.Results: (1)Expression of STAT1 and H2AX in TE1, TE13 and Eca109 cells were observed in level of mRNA and protein, but expression of STAT1 protein were not measured in TE13 and ECA109 with immunohistochemistry assay. (2) D0 value were 2.35Gy and 3.04Gy and SF2 value were 0.752 and 0.796 respectively with clonegenetic assay in TE13 and Eca109 cells, so radiosensitivity is higher in TE13 cells than Eca109 cells. (3)The expression of pSTAT1 protein in TE13 and Eca109 cells were not any changed at 0.5h after irradiated by 0-10Gy, and during 0-24h after irradiated by 4Gy. (4)Level ofγ-H2AX expression was markedly increased at 0.5h after irradiation by 0-10Gy in TE13 and Eca109 cells, and during 0-24h after irradiated by 4Gy. γ-H2AX protein in TE13 and Eca109 cells arrived at peak about 0.5h later, decreased at 1h and recovered to normal level at 24h after radiated by 4Gy. (5) H2AX nuclear foci were clearly present within 5 min after IR. The average number of H2AX-positive foci per cell increased at 0.5h after irradiation by 0-10Gy in TE13 and Eca109 cells, and during 0-24h after irradiated by 4Gy, and peaked 30 min after irradiation. Gradually, the number of H2AX-positive foci decreased to background levels within 24h. The average number of pSTAT1 nuclear foci were not formatted after irradiated, while the pSTAT1 nuclear foci were formatted after treated with cisplatin, but the average number of pSTAT1 nuclear foci was not any changed after irradiation and treated with cisplatin.Conclusion: (1) Expression of STAT1 and H2AX in different type of esophageal carcinoma cells protein were universally existed in level of mRNA and protein. (2) Radiosensitivity is higher in TE13 cells than Eca109 cells. (3) Irradiation can not affect the expression of STAT1 protein and the number of gene H2AX nuclear foci in esophageal cancer cells. the STAT1 nuclear foci was formatted after treated with cisplatin, but no changes of number of nuclear foci with irradiated. (4) The expression of H2AX protein and the number of gene H2AX nuclear foci have dose-effects relationship between ionizing radiation in esophageal cancer cells.更多还原