启动子甲基化致opcml失活在胃癌发病中的作用

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1、广州医学院硕士学位论文广州医学院硕士研究生学位论文启动子甲基化致OPCML失活在胃癌发病中的作用PromotermethylationtolossofOPCMLexpressioninprogressionofgastriccancer专业名称：病理学与病理生理学学位申请人：清导师：刘超教授广州医学院²广州二O一二年五月1广州医学院硕士学位论文目录中文摘要„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„1英文摘要„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„5中英文词汇对照

2、表„„„„„„„„„„„„„„„„„„„„„„„„„6前言„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„7第一部分OPCML基因在胃癌细胞株中的失表达及基因启动子CpG岛甲基化一、材料和方法„„„„„„„„„„„„„„„„„„„„„„„„„„„11二、结果„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„21讨论„„„„„„„„„„„„„„„„„„„„„„„„„„„„„22第二部分OPCML基因的功能学研究一、材料和方法„„„„„„„„„„„„„„„„„„„„„„„„„„

3、„24二、结果„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„37讨论„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„39结论„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„41参考文献„„„„„„„„„„„„„„„„„„„„„„„„„„„„„42致谢„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„47学位论文原创性说明„„„„„„„„„„„„„„„„„„„„„„„„482广州医学院硕士学位论文启动子甲基化致OPCML失活在胃癌发病中的作用研究生

4、：导师：中文摘要研究目的：探讨OPCML基因在胃癌细胞株中的失表达及其与基因启动子CpG岛甲基化的关系，并分析OPCML对胃癌细胞增殖的影响。方法:RT-PCR检测OPCMLmRNA在胃癌细胞系(SGC7901，AGS，MKN28，MKN45,N87，KATOIII，SNU1)的表达。甲基化特异性PCR(MSP)法检测胃癌细胞系中OPCML甲基化情况。5-AZA(5′-aza-2’-deoxycytidine)的去甲基化处理观察OPCML的表达变化。RT-PCR检测转染OPCML表达载体及pcDNA

5、3.1空载体后mRNA的表达情况。用MTT方法观察OPCML对AGS细胞活力的影响。集落形成实验法观察OPCML对胃癌细胞AGS集落形成的影响。OPCML表达载体转染HEK293A，AGS，MKN45细胞后，westernblot检测基因OPCML蛋白的表达。结果:(1)在胃癌细胞株中OPCMLmRNA的表达率显著低于正常胃组织。(2)胃癌细胞AGS和MKN45的甲基化程度明显高于正常胃组织中。(3)5-AZA处理MKN45细胞，OPCML表达上调。(4)OPCML表达载体转染HEK293A，AGS

6、，M45细胞后，RT-PCR检测显示高表达。(5)与转染空质粒的胃癌细胞相比，转染了OPCML的AGS和MKN45细胞活性明显降低。(6)外源性表达OPCML可抑制胃癌细胞AGS的集落形成率。(7)OPCML表达载体转染HEK293A，MKN45细胞后，Westernblot检测OPCML蛋白未见表达。关键词:胃癌细胞株;OPCML基因;甲基化3广州医学院硕士学位论文PromotermethylationtolossofOPCMLexpressioninprogressionofgastriccan

7、cerCandidateformaster:SupervisorProfessor:AbstractObjective：TodiscussthelossofOPCMLexpressioningastriccancercells,anditsassociationwithmethylationofpromoterCpGislands,andtoanalyzetheproliferationofectopicexpressionofOPCMLingastriccancercells.Methods：OP

8、CMLexpressionwasdetectedinhumangastriccancercelllinesbyRT-PCR.weanalyzedOPCMLgenemethylationusingMSP.OPCMLexpressioninresponsetodemethylationwasdetectedbyRT-PCR.Thecellviabilityassaywasperformedbymononuclearcelldireccytotoxicityassay(MT