【作者】 崔兴；

【导师】 徐瑞荣；

【作者基本信息】 山东中医药大学 ， 中西医结合临床， 2011， 博士

【摘要】 目的：研究肾阴虚型慢性再生障碍性贫血(CAA)患者线粒体突变情况和骨髓中ID4基因启动子区甲基化状态,探讨母系遗传的本质——线粒体和表观遗传学与肾阴虚型慢性再障发生、发展的关系,以期进一步研究慢性再障的发病机制。方法：收集8例诊断明确的肾阴虚型CAA患者骨髓和口腔黏膜上皮,另收集2例健康人骨髓做正常对照(仅用于ID4基因启动子区甲基化的对照),提取DNA。应用甲基化特异性聚合酶链反应(MS-PCR)检测ID4基因启动子区甲基化状态；进行线粒体DNA的全测序,在骨髓标本中与剑桥序列不一致的位点再与同一患者的口腔上皮正常组织的序列比较,若此位点的变化在口腔上皮组织中未出现,则定义为突变；若在骨髓标本和口腔上皮中均出现,则定义为多态。从中西医两种医学的母系遗传角度探讨其发病机制,并对结果进行分析。结果：(1)线粒体全测序表明许多患者的突变位点发生在与线粒体氧化呼吸链密切相关的区域,涵盖了了ND1-5、ND4L、ATP6、CYTB等多个线粒体DNA的编码基因。而很多的DNA多态性则位于D-loop区。(2)8名CAA患者均检测到了ID4基因的甲基化。结论：(1)线粒体呼吸链酶复合体表达水平的改变,可能导致线粒体呼吸链氧化磷酸化解偶联、“电子漏”产生ROS引起氧化应激和能量代谢障碍,这些变化共同参与造血干细胞衰竭的发生发展过程。因此,线粒体基因突变导致的呼吸链功能障碍可能在慢性再障的发病过程中起重要作用。(2)在所有患者骨髓细胞中均检测到了ID4基因甲基化,表观遗传学在CAA的发病机制中起到一定的作用,有助于慢性再障发病机制的进一步研究。(3)本研究从中西医的肾虚和母系遗传、表观遗传学两个方面初步探讨了慢性再障的发病机制,在国际上尚未见到类似研究,意义重大。更多还原

【Abstract】 Objective:This study was purposed to analyze the mitochondrial genome by gene amplification and investigate the difference of Id4 gene promoter methylation between healthy individuals and chronic aplastic anemia(CAA) patients suffer from kidney yin deficiency, thereby to investigate it’s meaning in the morbidity and progression of the disease.Methods:8 patients with CAA suffer from kidney yin deficiency including 3 males and 5 females. These samples represented by bone marrow which was anticoagulated by natrium citricum cells, and their oral epithelium were also collected for normal tissue comparison. (1)MS-PCR methods were used to detect the status of Id4 gene methylation in bone marrow of 2 healthy individuals and CAA patients. (2) Genomic DNA was extracted with an equal volume of phenol-chloroform. Mitochondrial genome was amplified by PCR. Then PCR products were gel purified and transferred to plasmid by T carrier. Whereafter the liquid that includes the aim segment were sent to sequencing. Results were compared with concomitantly studied normal tissues as well as mtDNA sequences in a standard database in revised Cambridge reference sequence (rCRS). Nucleotide changes that were both present in bone marrow and oral epithelium of the same patient were counted as polymorphisms.Results:(1) In our research, we found there were higher mutational rate in ND1-5、ND4L、ATP6、CYTB gene.And we found there were a lot of mitochondrial DNA polymorphisms in D-LOOP.(2) The results showed that Id4gene was unmethylated in bone marrow samples from healthy individuals, but Id4 gene methylation was found in all 8cases of CAA patients. Conclusion:(1) We are led to conclude that gene mutation can change the expression of Respiratory chain enzyme complex in CAA patients, which results in uncoupling of oxidative phosphorylation in mitochondrial respiration chain, oxidative stress caused by ROS from "electron leakage", energy metabolism impairment, this indicated that these changes may participate in the physiological and pathology processes of hematopoietic failure. Functional impairment of mitochondrial respiration chain induced by gene mutation may be a important reason of hematopoietic failure in CAA. (2) It is concluded thatas compared with healthy individuals, Id4gene in CAA patients was methylated in different degrees. The change of Id4 gene methylation is thought to be associated with occurence of CAA.更多还原