【作者】 姜洪华；

【导师】 周福生；

【作者基本信息】 广州中医药大学 ， 中西医结合消化， 2007， 博士

【摘要】 目的：对脾气虚证患者十二指肠粘膜线粒体DNA(mtDNA)全序列与线粒体超微结构进行初步探讨，了解脾气虚证与线粒体DNA多态性的相关性。方法：参照已经界定的排除标准，选择病例；分脾气虚证组、湿热证对照组和正常对照组；对十二指肠粘膜的超微结构在电镜下进行观察；提取并扩增线粒体DNA，行mtDNA全序列测定，对结果先BLAST，与剑桥序列进行比对，记录突变位点、突变种类、突变性质、合成氨基酸的密码子改变情况，利用Olige6.0软件进行多基因序列比对分析，了解线粒体的多态性。结果：1、脾气虚证和湿热证线粒体的结构均有不同程度的改变；其中同一种疾病，脾气虚证组与湿热证组线粒体结构的改变无明显差异：而同一种证型，慢性浅表性胃炎组与十二指肠球部溃疡组线粒体的改变差异明显，慢性浅表性胃炎组线粒体的改变较轻，十二指肠球部溃疡组线粒体的改变明显；脾气虚证十二指肠球部溃疡组的线粒体有明显的肿胀，变形，嵴减少或消失，有些线粒体有空泡形成，湿热证球部溃疡组线粒体也有肿胀、变形、嵴减少或消失等改变，还有些聚集在一起。2、对11个标本进行了mtDNA全序列重叠测序，测出率达99％，每个序列分正向和反向测序两次，有些序列多次重复检测，结果稳定。3、重叠测序后共检测出碱基突变470个。突变类型有：单个碱基突变和多个碱基突变，每种突变又包括碱基置换、碱基插入、碱基缺失：其中D-LOOP区断共64个，t—RNA区段共53个，r—RNA段共77个，CDS段共276个。脾气虚证组和慢性浅表性胃炎在D-LOOP区突变率高于其他组，脾气虚证和湿热证十二指肠球部溃疡组缺失突变高于其他组。4、移码突变共107个，碱基置换突变共314个(包括同义突变83个，错义突变261个，无义突变15个)。脾气虚证在同义、错义、无义和移码突变均等同或低于湿热证；湿热证在错义和移码突变中突变数明显增高；慢性浅表性胃炎组在同义突变中突变多，球部溃疡组在错义、无义和移码突变中突变多于其他组；脾气虚证组在转换、颠换方面均低于湿热证组；脾气虚证和湿热证球部溃疡组在插缺方面突变明显高于其他组。结论：1、同一证型(脾气虚证)不同病种(慢性浅表性胃炎和十二指肠球部溃疡)线粒体超微结构的改变不相同；2、脾气虚证mtDNA序列存在多态性改变；3、脾气虚证慢性浅表性胃炎组在D-LOOP区突变率高，可能是脾气虚证的分子基础；4、脾气虚证和湿热证的十二指肠球部溃疡组插入和缺失突变明显高于慢浅胃组和正常组，可能是十二指肠球部溃疡的分子基础。更多还原

【Abstract】 Aim: To explore the whole genome sequence and ultrastructure ofmitochondrion in duodenum mucosa from spleen qi-deficiency syndrome’s patient.To understand the relations between spleen qi-deficiency and polymorphismsof mitochondrial DNA (mtDNA).Method: Refer to having bordered the exclusive standard to choose caseswhich are divided into spleen qi-deficiency syndrome group、the wet and hotsyndrome group and normal contrastive group.By electron microscope to observethe ultrastructure of mitochondrial in duodenum mucosa. To extract andextend-prolifer mtDNA, to blast the result, to contrast with Cambridgesequence. Record the mutation Site、type、character and cipher code whichsynthesize amino acid. To understand the mtDNA’s polymorphisms, make use ofOlige 6.0 software to analyse the multi-gene sequence.Result: 1 There are different degree’s change in all groups. Nodifference is found between Spleen qi-deficiency syndrome and the wet and hotsyndrome group with same disease; there are obvious different between chronicshallow gastritis(CSG) and duodenum ulcer with same syndrome; CSG’s changesare lighter and duodenum ulcer’s changes are obviously more. Somemitochondrion swells, metamorphosis, which ridge become less, even vanish forduodenum ulcer of Spleen qi-deficiency syndrome, Some mitochondrion becomehollow; the wet and hot syndrome group’s changes are like this, somemitochondrion get together. 2 The eleven samples are surveyed for the wholemtDNA sequence, the complete rate is 99%; every sequence is measured frompositive and negative two directions, some are several times, the result issteady. 3 There are 470 mutation after repeat measure. The mutation typeshave single and multi-nucleotide mutation, every mutation still includeexchange、insert and defect, there are 64 mutations in D-LOOP section, 53 mutations in t-RNA section, 77 mutations in r-RNA section, 276 mutations inCD section. The mutations are more in D-LOOP section from Spleenqi-deficiency syndrome and CSG. The defect mutations are more in duodenum ulcer.4 The alter code’s mutations are 107, nucleotide exchange mutations are 314(same meaning mutations are 83, fault meaning mutations are 26, losing meaningmutations are 15), same meaning mutations、fault meaning mutations、losingmeaning mutations and alter code’s mutations are less in Spleen qi-deficiencysyndrome than other’s groups; fault meaning mutations and alter code’smutations are more in the wet and hot syndrome group than other’s groups;same meaning mutations are more in CSG; fault meaning mutations、losingmeaning mutations and alter code’s mutations are more in duodenum ulcer;transition and transversion are less in Spleen qi-deficiency syndrome group,insert and defect mutations are obviously more in duodenum ulcer group thanCSG group.Conclusion: 1 The ultrastructure of mitochondrion are different fordifferent disease(CSG and duodenum ulcer ) with same syndrome (Spleenqi-deficiency syndrome or the wet and hot syndrome group); 2 Spleenqi-deficiency syndrome’s mtDNA exist polymorphism; 3 The mutations are morein D-LOOP section from Spleen qi-deficiency syndrome, which is probably itsmolecule base. 4 Insert and defect mutations are obviously more in duodenumulcer group of Spleen qi-deficiency syndrome or the wet and hot syndrome group,which is probably its molecule base.更多还原