维吾尔族CYP21A2氧合活性对NAFLD线粒体功能影响机制研究

NAFLD/NASH, a manifestation of metabolic syndrome in the liver, has been considered as a mitochondrial disease, since mitochondrial dysfunction is involved at all successive steps leading to liver steatosis and NASH. In fat-engorged hepatocytes, several vicious cycles and chemically reactive lipid peroxidation products alter mitochondrial respiratory chain polypeptides and mitochondrial DNA to partially block the flow of electrons in the respiratory chain. To some extent, liver tissue of NAFLD is a model for mitochondrial dysfunction. Mitochondrial play a major role in fuel oxidation. The conversion of ATP formation and cholesterol synthesis is associated with the NADH: O2 oxidoreductase activity. It is well documented that hypoxia is an important pathogenesis factor for NAFLD development, but the molecular regulation of oxygen taking part in mitochondrial respiratory chain is not well understood. According to computer-based analysis of protein structure and biological function, the hemoglobin ring of 21-hydroxylase enzyme CYP21A2 is more like a regulator of energy conversion switch through controlling oxygenase activity of oxidative phosphorylation in mitochondrial. Our previous experiments indicated that the steroid 21 hydroxylase CYP21A2 gene expression in hepatocyte is significantly upregulated by oxygen - ion in NAFLD model animals. Computer analysis and our experiment research revealed the association of CYP21A2 gene with oxygen that lead to the following scientific hypothesis: hepatic CYP21A2 gene may affect mitochondrial respiratory chain activity by regulating oxygen - ion binding to mitochondrial respiratory chain. To verify the hypothesis, HepG2 cells lines of CYP21A2 gene intervened expression is planed to explore CYP21A2 gene function, mitochondrial oxidative phosphorylation synthesis, cholesterol synthesis, insulin signaling pathway, hypoxia-inducible factor, miR-210, and micro- RNA (miR-122).The correlation between CYP21A2 gene encoded the steroid 21 hydroxylase oxygenation activity and mitochondrial energy transformation will be analyzed. CYP21A2 gene expression and polymorphisms on human hepatocytes and white blood cells are examined and compared between NAFLD and healthy control, respectively. Mitochondrial oxidative phosphorylation activity, cholesterol synthesis, insulin signaling pathways, CYP21A2 expression levels and gene polymorphisms in Xinjiang Uygur NAFLD patients and control liver tissue will be examined and compared. Then, peripheral blood genomic CYP21A2 gene polymorphisms will be examined and compared between Uygur NAFLD patients and healthy controls. The elucidation of the correlation between CYP21A2 gene oxygenation activity and mitochondrial respiratory chain activity of NAFLD will add our knowledge to the pathogenesis mechanism of hypoxia in the metabolic syndrome.

缺氧与NAFLD发生密切相关，NAFLD肝细胞线粒体存在呼吸功能障碍，是研究缺氧与线粒体呼吸障碍理想模型。氧分子参与线粒体氧化磷酸化呼吸链调控相关研究很少；课题组基于前期实验和计算机蛋白质构象及功能分析，推测肝脏CYP21A2基因编码血色素环氧合活性可能参与肝脏线粒体呼吸链活性调控，课题组拟从细胞系CYP21A2功能干预表达、NAFLD肝组织与正常肝组织CYP21A2表达水平、基因多态性分布比较，从线粒体呼吸链活性、胆固醇合成、胰岛素信号通路、低氧调控基因和microRNA表达水平角度探讨CYP21A2氧合活性对线粒体呼吸链活性影响；采用新疆维吾尔族NAFLD患者和正常对照CYP21A2外周血基因多态性分布差异，探讨CYP21A2氧合活性与NAFLD发生的相关性， 从疾病角度研究CYP21A2氧合活性对肝脏线粒体呼吸链活性影响在物质-能量代谢中的作用，有助于探索缺氧对人体物质能量代谢影响。

CYP21A2基因缺陷引起21一羟化酶缺乏症导致先天性肾上腺皮质增生症，是常染色体隐性遗传疾病，与导致遗传性库兴综合征。CYP21A2仅在肝脏细胞和肾上腺表达。.本项基金研究是基于一篇采用射线衍射探讨蛋白质空间结构与功能的研究，该研究核心观点是：CYP21A2基因的血色素环存在空间上的大与小的区别，该项研究认为这将影响到该基因相关蛋白血色素分子与氧分子结合的效率，而这种氧结合效率可能对细胞内线粒体内能量转换的核心环节——发生的氧化磷酸化产生影响，这篇文献没有进一步在细胞系水平展开研究以证实作者的观点，未见探讨肝脏CYP21A2氧合作用与肝细胞线粒体氧化磷酸化的研究。.通过细胞水平研究CYP21A2的氧合活性是否对肝脏细胞线粒体氧化磷酸化过程发生影响。我们采用动物模型，高氧干预，肝硬化与肝癌组织、全基因组表达谱，对 HepG2细胞迁移与侵袭作用、细胞周期、细胞活力、细胞凋亡等研究，观察CYP21A2在外源性氧供应差异和基因表达差异之间的肝细胞功能性变化。.肝组织的CYP21A2表达水平，无论是模式动物、人体肝组织的肝硬化阶段和肝癌阶段，表达水平异常稳定，没有因为外源性提高氧的供应量，也未因为肝细胞呼吸链而发生改变。CYP21A2基因芯片表达谱测序，选取30个目的基因。采用细胞转染技术， CYP21A2过表达时，基因表达水平升高200倍，沉默时基因表达水平下降约80%，萤光定量检测发现有16个基因与芯片表达谱结果一致。CYP21A2基因对 HepG2细胞迁移与侵袭作用、细胞周期G0/G1产生显著性影响，对HepG2细胞活力、细胞凋亡无影响。.肝细胞的能量-物质代谢出现了非常显著性的差异，表现为蛋白质合成减少、胆固醇合成基因表达增加，线粒体呼吸链电子传递核心NADH系统受到显著性影响。这些肝细胞系表现出来的变化：蛋白质合成减少、胰岛素抵抗增加、氧化磷酸化水平下降、胆固醇合成增加，细胞的三羧酸循环呼吸链关键酶表达下降。关闭CYP21A2上的血色素环可以上调缺氧耐受基因HIF1A的表达水平；EGFR、GPC3这个与恶性肿瘤密切相关的基因表达水平也大幅提高，这似乎提示该基因可能成为肝细胞肝癌治疗的一个潜在靶点。这些实验提示CYP21A2在肝脏细胞的功能的确是与细胞氧摄入功能相关，这是目前首次发现CYP21A2在肝脏细胞的功能.