【作者】 吴进峰；

【导师】 沈薇；

【作者基本信息】 重庆医科大学 ， 内科学， 2010， 博士

【摘要】 研究背景及目的MICA/B是固有免疫系统的组成部分,在包括肝癌在内的多种肿瘤中高表达,通过与其受体NKG2D相互作用,发挥肿瘤免疫作用。脱氧氮杂胞苷5-aza-dC是一种甲基化酶抑制剂,我们以往的实验发现5-aza-dC可诱导肿瘤细胞MICB的表达,并增强NK细胞对肿瘤细胞的杀伤作用。最新研究表明RNAi机制也与固有免疫系统相关,Dicer是RNAi系统中的关键酶,在秀丽隐杆线虫中Dicer的缺失,可引起固有免疫基因表达的改变。为了研究MICA与肝癌的关系,本课题将首先以HepG2细胞为研究对象,观察MICA在L02细胞和HepG2细胞中的表达差异,并进一步研究Dcier缺失及5-aza-dC对HepG2细胞MICA的诱导作用及可能机制。最后我们还将从临床角度出发,观察MICA、Dicer在HCC患者中的表达是否异常并初步探讨其意义和机制。方法1.为了沉默Dicer,使用Dicer siRNA转染HepG2细胞;为了阻断ATM,使用ATM特异性抑制剂咖啡因处理HepG2细胞或ATM siRNA转染HepG2细胞。2. Realtime RT-PCR检测Dicer、ATM、MICA mRNA表达水平,流式细胞术检测细胞膜MICA蛋白水平。3.免疫荧光染色法检测γ-H2AX水平。4.收集经病理证实的36例HCC患者的肝癌、邻近非癌组织标本及患者临床资料(年龄、性别、肿瘤大小、肿瘤转移、肿瘤个数、肿瘤分期),Realtime RT-PCR检测肝癌及癌旁组织MICA、Dicer mRNA表达水平。结果1.流式细胞术结果显示HepG2细胞高水平表达MICA,而正常肝细胞L02几乎不表达MICA。2. 5-aza-dC处理或选择性沉默Dicer可上调HepG2细胞MICA的表达(P <0.05),并引起DNA损伤特异性指标γ-H2AX增高。3. 5-aza-dC或选择性沉默Dicer上调HepG2细胞MICA作用可被ATM特异性阻滞剂咖啡因或ATM特异的siRNA所阻断(P <0.05)。4.Realtime PCR结果显示MICA mRNA在肝癌标本及邻近非癌标本中表达水平无显著差异(P >0.05)。而Dicer mRNA在肝癌标本中的表达低于邻近非癌标本,差异有显著性(P<0.05)。5. Spearman’s rank-order coefficients统计分析表明肝癌标本中MICA、Dicer mRNA水平与患者年龄、性别、肿瘤大小、肿瘤转移、肿瘤个数、肿瘤分期等临床参数无显著相关性(P >0.05)。结论1. 5-aza-dC可诱导HepG2细胞MICA的表达,其机制可能与5-aza-dC引起的ATM依赖的DNA损伤途径有关。2.Dicer在肝癌中低表达,提示RNAi系统可能与肝癌的发生发展相关。Dicer的缺失可能继发性启动ATM依赖的DNA损伤途径并激活内源性免疫系统。更多还原

【Abstract】 OBJECTIVEMICA and MICB, as components of innate immune system, play a role in tumor immune surveillance via interation with NKG2D.Our previous study indicated that 5-aza-dC ,a kind of methylase inhibitor ,induced MICB expression in a DNA damage-dependent manner, which in turn sensitized tumor cells to NKL-cell-mediated lysis. RNAi acts constitutively to silence the innate immune response, and innate immunity genes are misregulated in Dicer-deficient Caenorhabditis elegans. Here we will determine the expression level of MICA in HepG2 cells (an HCC cell line) and L02 cells ( a normal liver cell) , and investigate the effect of 5-aza-dC or Dicer knockdown on MICA expression in HepG2 cells. Furthermore, we will detect the expressions of MICA and Dicer in HCC.METHODS1. HepG2 cells were treated with 5-aza-dC, caffeine , ATM-specific siRNA or Dicer-specific siRNA. The cell surface MICA protein on HepG2 and L02 cells were determined using flow cytometry. The mRNA levels were detected using real time RT-PCR. Theγ-H2AX levels were examined by Immuno?uorescence microscopy.2. The mRNA levels of MICA and Dicer in 36 paired cancerous and corresponding adjacent non-neoplastic tissues from 36 HCC patients undergoing surgery were detected using real time RT-PCR. Furthermore, the mRNA levels of MICA and Dicer in cancerous tissues were analyzed respectively to clinical features including age, sex, tumor size, tumor metastasis status, tumor number and stage.RESULTS1.MICA was undetectable on the surface of L02 cells, but was highly expressed on HepG2 cells. MICA expression in HepG2 cell was upregulated in response to 5-aza-dC treatment or Dicer siRNA , and the upregulation of MICA was partially prevented by pharmacological or genetic inhibition of ataxia telangiectasia mutated (ATM) kinase.2.Dicer mRNA level was significantly lower in malignant tissues than in the corresponding non-neoplastic tissues in 36 HCC patients. There was no significance on MICA mRNA levels between malignant tissues and the corresponding non-neoplastic tissues in 36 HCC patients. Neither the Dicer nor MICA level was associated with clinical characteristics including age, sex, tumor number, tumor size, tumor stage, or distant metastasis in HCC cases.CONCLUSIONSOur data suggest that 5-aza-dC or Dicer loss induces the expression of MICA by a ATM-dependent DNA damage pathway, and RNAi mechanism may contribute to innate immune system on tumor immune surveillance.更多还原