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目的探讨内质网氧化还原酶-1α(endoplasmic reticulum oxidoreductin 1-α,ERO1α)及其DNA甲基化在同型半胱氨酸(homocysteine,Hcy)抑制肝细胞增殖中的作用。方法以0μmol·L-1Hcy为正常对照组(NC group),以100μmol·L-1Hcy为干预组(Hcy group),干预肝细胞48h后,四甲基偶氮唑盐比色法(MTT)检测肝细胞增殖活力的变化;实时定量聚合酶链反应(qRT-PCR)及Western blot分别检测ERO1αmRNA和蛋白表达水平;构建ERO1α真核表达质粒,转染肝细胞后,荧光显微镜下观察转染效率并以qRTPCR及Western blot验证ERO1α是否过表达,后用MTT法检测肝细胞增殖活力的变化;巢式降落式甲基化特异性PCR(nt MS-PCR)检测ERO1αDNA甲基化水平。结果与正常对照组相比,Hcy干预组肝细胞内ERO1αmRNA及蛋白表达水平降低,且细胞增殖活力减弱(P<0.01)。测序结果证明ERO1α重组质粒构建成功,荧光显微镜下可见绿色荧光蛋白大量表达,qRT-PCR及Western blot验证结果显示ERO1α过表达(P<0.01),而MTT法结果表明ERO1α过表达可缓解Hcy导致的肝细胞增殖抑制(P<0.01)。Hcy刺激后ERO1αDNA甲基化水平升高(P<0.05)。结论 Hcy通过下调ERO1α表达抑制肝细胞增殖,而ERO1α启动子区DNA甲基化是其重要的调控机制。
Objective To investigate the role of endoplasmic reticulum oxidoreductin 1-α (ERO1α) and its DNA methylation in the inhibition of hepatocellular proliferation by homocysteine (Hcy). Methods 0μmol·L-1Hcy was used as the normal control group (NC group) and 100μmol·L-1Hcy was used as the intervention group (Hcy group). After intervention of the hepatocytes for 48h, MTT assay The expression of ERO1αmRNA and protein were detected by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot respectively. ERO1α eukaryotic expression plasmid was constructed and transfected into hepatocytes. The transfection efficiency was observed by fluorescence microscopy ERO1α was overexpressed by qRTPCR and Western blot. The proliferation of hepatocytes was detected by MTT assay. The methylation level of ERO1α was detected by nested-type-specific methylation-specific PCR (nt MS-PCR). Results Compared with the normal control group, the expression level of ERO1α mRNA and protein in Hcy intervention group decreased and the cell proliferation activity decreased (P <0.01). The results of sequencing showed that the ERO1α recombinant plasmid was successfully constructed and the expression of green fluorescent protein was abundant under fluorescence microscope. The results of qRT-PCR and Western blot showed that ERO1α was overexpressed (P <0.01), while MTT assay showed that ERO1α overexpression could alleviate Hcy-induced Inhibition of hepatocyte proliferation (P <0.01). ERO1α DNA methylation levels increased after Hcy stimulation (P <0.05). Conclusion Hcy inhibits the proliferation of hepatocytes by down-regulating the expression of ERO1α, while DNA methylation in the promoter region of ERO1α is an important regulatory mechanism.