摘要
目的观察主要组织相容性复合体Ⅰ类分子A（MICA）在HepG2与L02细胞株中表达的差异及脱氧氮杂胞苷（5-aza-dC）对HepG2细胞株MICA表达的影响，探讨毛细血管扩张眭共济失调突变蛋白（ATM）依赖的DNA损伤途径与5-aza-dC调节MICA表达的关系。方法同时接种并培养L02和HeDG2细胞，在同一时间点收取细胞，流式细胞仪检测细胞膜MICA蛋白表达水平；不同浓度5-aza—dC（0．1、1．0、5．0mmol／L）作用于HepG2细胞不同时间（24、48、72、96h）后，定量PCR检测MICA mRNA水平以寻找5-aza-dC最佳作用浓度和时间；ATM特异性抑制剂咖啡因或RNA干扰技术干扰ATM表达，定量PCR检测细胞mRNA水平，流式细胞术检测细胞膜MICA蛋白表达水平。结果数据比较采用Kruskal-Wallis和Mean-Whitney U方差分析。结果流式细胞结果显示，HepG2细胞高水平表达MICA，而正常肝细胞L02几乎不表达MICA；5-aza—dC处理可上调HepG2细胞MICA的表达（x^2=7．20，P〈0．05），这种上调作用可被ATM特异性阻滞剂咖啡因和ATM特异的小分子干扰RNA所阻断（U=0．00，P〈0．05）。结论MICA在正常肝细胞株中无表达，在肝癌细胞株中高表达；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。
Objective Major histocompatibility complex class I＂Crelated molecules A and B （MICA and MICB） are innate immune system ligands for the NKG2D receptor expressed by natural killer cells and activated CD8（＋）T cells. Our previous study showed that 5-aza-2＇-deoxycytidine（5-aza-dC）, a DNA methyltransferase inhibitor, can induce the expression of MICB and sensitized cells to NKL-cell-mediated cytolysis. The aim of this study was to determine the expression level of MICA in HepG2 cells （an HCC cell line） and L02 cells （ a normal liver cell）, and to investigate the effect of 5-aza-dC on MICA expression in HepG2 cells. Methods Cells were treated with 5-aza-dC, caffeine and ATM-specific siRNA. The cell surface MICA protein on HepG2 cells and L02 cells was determined using flow cytometry. The mRNA level was detected using real time RT-PCR. Result MICA was undetectable on the surface of L02 cells, but was highly expressed on HepG2 cells. MICA expression was upregulated in response to 5-aza-dC treatment （P 〈 0.05）, and the upregulation of MICA was partially prevented by pharmacological or genetic inhibition of ataxia telangiectasia mutated （ATM） kinase （P 〈 0.05）. Conclusion Our data suggest that 5-aza-dC induces the
expression of MICA by a DNA damage-dependent mechanism.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2009年第9期675-678,共4页
Chinese Journal of Hepatology
基金
国家自然科学基金（30700708、30871160）
重庆市自然科学基金（2007BB5308）
