从母系遗传及端粒衰老学说研究肾虚在慢性再障中的发病机制

The etiology and pathogenesis of chronic aplastic anemia is attributed to the deficiency of kidney.From the traditional Chinese medicine theory of "kidney is the congenital foundation", "Lord of renal hidden essence" and "kidney dominating bone marrow" ,we can found that "kidney essence"are closely related to the proliferation, differentiation, senescence and apoptosis of bone marrow cells. On the first part ,we sought to link the " Life comes from essence of parents " of "Huang Di Nei Jing" with the "Yin essence" of cingenital, and with the properties of mitochondrial DNA-the maternal genetic, we draw a conclusion that the "Yin essence" is the maternally inherited fundamental - - "mitochondria" .Recent studies have revealed, telomere aging theory in aplastic anemia bone marrow failure process played a crucial role, this is square with the notion of traditional Chinese medicine kidney aging theory . Based on the above two points, we design the topic..We found that there are many mutations of mitochondrial DNA in kidney yin deficiency group patients with chronic aplastic anemia.Therefore, in this paper,we are trying to detect the mitochondrial DNA mutations; the telomere length and the telomerase activity,to find the relations of mitochondrial mutations, telomere-telomerase and the TCM Syndrome Types, in order to find the relations of traditional Chinese medicine theory and molecular biology, and the relations of traditional Chinese medicine and the theoretical basis of molecular biology in chronic aplastic anemia.

慢性再生障碍性贫血中医病因病机多责之于"肾虚"，依据中医传统理论"肾为先天之本"、"肾主藏精"、"肾主骨生髓"，"肾精"与骨髓细胞增殖、分化、衰老乃至凋亡等基本生命活动相关。第一部分研究由《内经》中"以母为基"联系到了先天之"阴精"，并结合线粒体"母系遗传"、"能量工厂"的特性，得出了中医的"阴精"即是母系遗传的根本- - "线粒体"的论点。研究表明，端粒衰老学说在慢性再障骨髓衰竭过程中起到了至关重要的作用，这与中医的肾虚衰老学说遥相呼应。基于以上两点，设计了该课题。.课题组既往对慢性再障线粒体的研究发现，肾阴虚型患者发现许多突变位点。本课题拟通过收集肾阴虚、肾阳虚、肾阴阳两虚慢性再障患者骨髓，检测线粒体突变、电镜观察线粒体形态；检测端粒长度、端粒酶的活性；研究其与慢性再障中医辨证分型的相关性，以期寻找中医宏观理论与现代医学微观指标的结合点，并为中医药治疗慢性再障找到分子生物学水平的依据。

一、项目的背景.慢性再生障碍性贫血中医病因病机多责之于“肾虚”，中医传统理论的“肾为先天之本”、“肾主藏精”、“肾主骨生髓”，与骨髓细胞增殖、分化、衰老乃至凋亡等基本生命活动相关。分子生物学研究表明，线粒体突变和端粒衰老学说在再生障碍性贫血骨髓衰竭过程中起到了至关重要的作用，这与中医的肾虚衰老学说遥相呼应。.二、主要研究内容.通过检测线粒体突变、端粒长度、端粒酶活性分析、T淋巴细胞亚群、ID4基因甲基化情况、代谢组学及蛋白组学的差异，来进行分子生物学指标与慢性再障中医辨证分型相关性的研究。.三、重要结果.我们推测以上相关指标，在慢性再障骨髓衰竭的发生发展中起到了极其重要的作用。.四、关键数据.1. 线粒体全测序表明三组患者的突变位点均发生在与线粒体氧化呼吸链密切相关的区域。.2. 慢性再障患者端粒长度较正常对照明显缩短。而且这些患者的T/S值（端粒相对长度）的大小与该患者WBC、Hb、PLT水平呈正相关；在三组患者中，肾阴虚组患者T/S平均值最短，肾阴阳两虚组其次，而肾阳虚组T/S平均值最长。.3. 慢性再障患者CD4+/CD8+比值较正常对照明显降低。在三组患者中，肾阴虚组患者CD4+/CD8+比值平均值最小，而肾阳虚组CD4+/CD8+比值平均值最大，而且与T/S值（端粒相对长度）呈正相关性。.4. 有33例患者出现了ID4基因甲基化，其中肾阴虚组有12例（60% 阳性率），肾阳虚组有11例（55% 阳性率），肾阴阳两虚组有5例（25% 阳性率），而正常对照组中未能发现ID4基因甲基化情况。.5. 最终我们在再障患者血清中确定了23个有意义的代谢物，分别涉及Biosynthesis of amino acids、Aminoacyl-tRNA biosynthesis 、ATP-binding cassette transporters (ABC transporters )和 TCA cycle 代谢通路。.6. 再障患者TERT表达水平均较正常对照组明显降低（P＜0.05）。.7. 慢性再障患者血浆与正常人血浆中存在差异表达蛋白质，并且不同证型之间差异也比较明显。.五、科学意义.将慢性再障辨证分型与上述指标相结合来研究，寻找中医宏观理论与西医微观分子生物学指标的结合点。本研究能充实中医学的理论，并为中医药治疗慢性再障找到分子生物学依据。