1、叶酸代谢酶相关亚甲基四氢叶酸还原酶基因多态性与2型糖尿病及代谢综合征易感性的关联研究 2、膀胱癌石蜡包埋组织microRNA检测及临床意义的初步探讨

【作者】 陈爱荣；

【导师】 王志平；

【作者基本信息】 兰州大学 ， 细胞生物学， 2010， 博士

【摘要】 背景和研究目的代谢综合征(Metabolic Syndrome, MetS)及2型糖尿病(Type 2 diabetes mellitus,T2DM)是近十年来全球范围内发病率猛增的代谢性疾病,尤其是发展中国家,包括中国。随着居民饮食结构、生活方式的改变,人均期望寿命明显延长,我国T2DM及MetS的发生率和死亡率均呈逐年上升趋势。胰岛素抵抗(Insulin resistance, IR)目前被公认为是与肥胖相关的代谢性疾病主要的病理生理基础。MetS及T2DM都是受饮食及基因影响的复杂疾病。高同型半胱氨酸血症已被明确是动脉粥样硬化、冠心病及脑血管疾病的致病因素。造成高同型半胱氨酸血症(Hyperhomocysteinemia, Hhcy)的两个致病因素-饮食不充分导致叶酸及维生素B12缺乏及相关致病基因的突变也成为MetS和T2DM病因研究的热点。同时营养基因学的发展使得探究高同型半胱氨酸血症临床药学干预的新措施以降低代谢综合征、2型糖尿病及心脑血管疾病的发病危险性也成为广受重视的新课题。饮食不充分因影响叶酸和维生素B12的水平可导致高同型半胱氨酸血症。亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase, MTHFR)是甲硫氨酸—叶酸代谢中的关键酶,可将N5,N10—亚甲基四氢叶酸还原为N5-甲基四氢叶酸,成为体内叶酸的主要活性形式。同时使血液中同型半胱氨酸发生甲基化,生成甲硫氨酸。正常的MTHFR活性对于维持同型半胱氨酸代谢的平衡具有重要作用,MTHFR基因677位C→突变造成MTHFR活性降低可使同型半胱氨酸转变为甲硫氨酸的过程出现障碍,造成高同型半胱氨酸血症。这种突变已成为一种新的代谢性疾病的独立危险因素,特别是代谢综合征,2型糖尿病及胰岛素抵抗。病理及临床研究的结果表明,炎症在动脉粥样硬化的每一个阶段都具有重要的作用。炎症因子的增加,如高敏C反应蛋白是心血管疾病的风险增加的预测指标,并与动脉粥样硬化和胰岛素抵抗密切相关。目前关于亚甲基四氢叶酸C677T基因多态性和胰岛素抵抗相关性研究还未见报道。同时,联合研究N5,N10-亚甲基四氢叶酸还原酶基因多态性、高敏C反应蛋白及饮食叶酸,维生素B12因素和代谢综合征及胰岛素抵抗相关性在亚洲人群的研究更显重要。我们的研究目的是在中国人群中调查叶酸代谢相关的亚甲基四氢叶酸还原酶基因多态性,揭示亚甲基四氢叶酸还原酶基因多态性与胰岛素抵抗的关系及代谢综合征和2型糖尿病的易感性。并进一步分析基因和环境因素在胰岛素抵抗和代谢综合征相关疾病危险因素发展中的作用。材料和方法研究对象：纳入研究对象来自2007年1月至2008年1月兰州大学第二医院内分泌科,根据世界卫生组织(WHO)的诊断标准确诊的158例初发2型糖尿病。健康对照55例,来自常规体检,血糖正常((3.3-6.1 mmol/L),不伴其他代谢疾病如高血压,高脂血症和肥胖。所有对象都是中国汉族人群,排外其他炎症和肿瘤。近两周未服用过维生素。研究方法：在158例T2DM患者中伴根据中华医学会糖尿病学分会(CDS,Chinese diabetic society)关于MetS的诊断标准确定MetS者(118)和不伴MetS(40)及55例健康人群中采用聚合酶链-限制性片断长度多态性(PCR-RFLP)方法检测MTHFRC677T多态位点。包括血样收集和DNA的提取；MTHFR基因型的检测(MTHFR基因片段扩增；Hinf I酶切PCR产物；2%琼脂糖凝胶电泳分析鉴定基因型)。放射免疫法检测血清胰岛素,叶酸和维生素B12。高敏C反应蛋白检测用免疫比浊法。统计分析全部在SPSS16.0 for Windows和Microsoft Excel 2003软件中进行。结果1.158例T2DM,根据中华糖尿病学分会(CDS)提出的中国人群关于MS的诊断标准,结果118例确诊MetS,其余40人没有MetS。T2DM相关的MetS患病率在中国北方人群中为75%(118/158),其中,男性MetS患病为71.4%(70/98),而女性则高达80%(48/60)。2.T2DM患者共158例,年龄33-80岁。<50岁人群MetS患病率为65.7%(23/35),而≥50岁人群MetS患病率为76.4%(101/123)。MetS患病率随年龄而增长,年龄≥60岁的老人,MetS患病率高达74.1%(60/81),是<60岁人群MetS患病率35.1%(37/77)的近2倍。3.T2DM合并MetS组患者和正常对照组相比BMI, TCHO, TG, LDL, UA, FPG, SBP, DBP, HOMA-IR和hs-CRP显著增加(P<0.05)。和没有MetS的单纯T2DM患者相比,T2DM伴发MetS患者具有更高水平的TCHO, TG, UA, SBP, DBP和HOMA-IR(P<0.05)；同时叶酸和维生素B12水平显著降低(P<0.05)。另外有MetS的T2DM患者的BMI和hs-CRP水平也高于没有MetS患者,这些差异没有统计学意义。4.T2DM伴MetS组患者中,MTHFR基因TT型患者和CC型及CT型相比较具有显著升高的血清TG, hs-CRP (P<0.05)和UA及HOMA-IR(P<0.001)。相反,血清维生素B12水平在TT型患者中和CC及CT型相比较却显著降低(P<0.01)。而其余临床检测指标包括BMI, SBP, DBP, HDL和血清叶酸水平在三种基因型CC, CT, TT型之间没有差别。5.在T2DM伴MetS患者中,MTHFR基因纯和型TT和杂和型CT的频率分别是19.5%和51.7%。这些值不仅显著高于健康对照组7.3%和30.9%,也高于T2DM不伴MetS组10%和32.5%。MTHFR基因等位基因总T的频率在健康对照组为22.7%,在T2DM不伴MetS组为26.3%,在T2DM伴MetS组却显著增加到45.3%(x2=20.665,P<0.001)。结论1. MTHFR基因C677T多态性的基因可能参与中国汉族MetS人群胰岛素抵抗。2.MTHFR基因增加MetS TT基因型超敏-C反应蛋白,甘油三酯和降低血清中维生素B12的含量水平,可能和中国汉族MetS相关。MTHFR基因C677T在2型糖尿病伴MetS的发展中具有重要作用。3. MTHFR基因总的T基因型频率从2型糖尿病不伴MetS患者的26.3%显著增加到伴MetS患者的45.3%。4.T等位基因增加2型糖尿病患者患代谢综合征的易感性。背景和研究目的膀胱癌是泌尿系统中最常见的肿瘤,80%为尿路上皮癌。许多膀胱癌临床患者诊断出时已有远处转移,导致手术切除率低,预后差。因此,有必要对膀胱癌发病分子机理进行深入研究,探索新的早期诊断与治疗方法。微小核酸(microRNA, miRNA)是新发现的一种长度为22核苷酸的非编码单链小分子RNA,占人类基因1%,却调节着至少30%的mRNA,近年来,和肿瘤关系研究成为热点。miRNAs所起的作用类似于抑癌基因和癌基因的功能,目前,关于膀胱癌miRNA研究报道很少,尚未见到膀胱肿瘤石蜡包埋组织microRNA表达谱的报道。我们结合前期对膀胱癌研究的工作基础,以福尔马林固定石蜡包埋(formalin-fixed paraffin-embedded tissue, FFPE)组织为研究材料。对膀胱癌石蜡组织miRNAs分子进行检测。材料和方法103例膀胱癌组织取自兰州大学第二医院泌尿研究所存档的1998年-2000年及2004年-2005年间的组织蜡块。其他手术切除的膀胱正常组织,均福尔马林固定后石蜡包埋。采用miRNA基因芯片技术的miRNA表达谱的分析方法和PCR法,评价FFPEmiRNA表达谱分析的有效性和可行性。筛选与膀胱癌生物学特征密切相关的差异表达miRNA。结果1 microRNA不易降解,提取RNA可提供有效miRNA芯片检测的要求；2与正常膀胱组织相比,膀胱尿路上皮癌组织中有上调基因15个,分别为miR-141, miR-429, miR-200a, miR-205, miR-203, miR-200c, miR-190, miR-200b, miR-210, miR-10b, miR-425, miR-34a, miR-191, miR-31, miR-10a.下调基因为3个,miR-487a, miR-886-5p, miR-486-5p。没有发现和病理分期有关,变化倍数和分期间没有相关性。3基因芯片能有效区分正常膀胱组织及不同分期的肿瘤组织.4.FFPE膀胱癌组织miRNA-203较正常显著上调134倍,而miRNA-141较正常显著上调373倍。5 miRNA-200家族成员(miRNA-200a, miRNA-200b, miRNA-200c, miRNA-141和miRNA-429)膀胱癌组织较正常组织升高,miR-141, miR-429, miR-200a, miR-200b、miR-200c分别较正常上调337,131,21,18,10倍。结论1.石蜡包埋组织(FFPE)可作为检测microRNA的有效标本；是研究miRNA的重要样品资源；2. FFPE组织存放时间长短(10年期；5年期)均能有效检测miRNA3.基因芯片分析膀胱癌石蜡包埋组织和正常膀胱粘膜相比,以表达差异>3倍为标准,膀胱尿路上皮癌组织中有上调基因15个,分别为miR-141, miR-429, miR-200a,miR-205,miR-203, miR-200c, miR-190, miR-200b, miR-210, miR-10b, miR-425, miR-34a, miR-191, miR-31, miR-10a.下调基因为3个,miR-487a, miR-886-5p, miR-486-5p。4. FFPE膀胱癌组织miRNA-203较正常显著上调134倍,而miRNA-141较正常显著上调373倍。故推测miRNA-203、miRNA-141可应用于膀胱尿路上皮癌的诊断判断。5. microRNA变化和病理分期无相关。6. miRNA-200家族成员(miRNA-200a, miRNA-200b, miRNA-200c, miRNA-141和miRNA-429)参与膀胱癌的发生。更多还原

【Abstract】 Background and ObjectiveMetabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM) are increasing metabolic disease in recent years in the whole whold especially in our developing country, including in China.Their incidence rate and mortality rate show a yearly increasing trend with changes in dietary structures and life styles of residents and prolongation of percapita expectancy in last decades. Insulin resistance is the most important pathological bases of obesity related metabolic disease.Metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM) are complex diseases that are affected by dietary intake and genetics. Hyperhomocysteinemia (Hhcy) has been proved is a risk factor for atherosclerosis and cardiovascular diseases. Two risk factors-lack of folic acid metabolism related diet and related genes mutations of Hhcy are becoming the high light cause research in MetS and T2DM. At the same time the development of nutrigenomics allows exploration of Hhcy clinical pharmacy intervention initiatives to reduce the metabolic syndrome, type 2 diabetes and cardiovascular diseases risk has become a widely important new topics.Dietary inadequacy can result in HHcy by presumably affecting the level of folate and vitamin B12. Methylenetetrahydrofolate reductase (MTHFR) is a key regulator for concentration of homocystine. MTHFR can irreversibly convert 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which is the predominant form of folate found circulating in the blood and is the methyl donor during Hey remethylation. In addition, the activity of MTHFR is important for metabolism homeostasis. The C to T mutation of the MTHFR gene at nucleotide position 677 (MTHFR677>T) decreases its enzymatic activity, leading to accumulation of Hey. Such a mutation has emerged as a novel independent risk factor for metabolic disease, especially in Met,T2DM.The results of pathological and clinical studies suggest that inflammation plays a prominent role at every stage of atherogenesis. Increased levels of inflammatory proteins, such as hs-CRP, are predictive markers of an increased risk of cardiovascular diseases and are associated with atherosclerotic disease and insulin resistance.The relationship between the MTHFR C677T gene polymorphism and insulin resistance has not been previously reported. The association between MetS and the N5, N10-methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism, high-sensitivity C-reactive protein (hs-CRP) and the dietary components folate and vitamin B12 in Asians has not been well characterized. The purpose of our study is to explore distributions of MTHFR gene polymorphisms of folate metabolic enzymes in a Chinese population and associations between these polymorphisms and insulin resistance and susceptibility to Met and T2DM, and further to analyze the role of gene and environment interactions in the development of insulin resistance and risk factors of MetS.Materials and MethodsA total of 158 T2DM patients and 55 healthy subjects were enrolled in this study. The patients were enrolled in the department of Endocrinology of the Second Hospital of Lanzhou University between January 2007 and January 2008 were diagnosed with T2DM according to the criteria of the World Health Organization (WHO). The healthy participants were recruited during routine health screenings, had a normal glycemic index (3.3-6.1 mmol/L) and did not have metabolic diseases, such as hypertension, hyperlipidemia and obesity. All the subjects were of Han Chinese origin and did not have inflammatory diseases or cancers of any type.The MTHFR C677T gene polymorphism were analyzed by Polymerase Chain Reaction-Restriction Fragment Lenth Polymorphism (PCR-RFLP) in newly diagnosed T2DM patients with (118) and without MS (40) and in healthy subjects (55). Levels of insulin, folate and vitamin B12 by radioimmunoassay and of hs-CRP by immunoturbidimetry also were measured.All statistical analysis was processed by SPSS 13.0 for windows and Microsoft Excel 2003.Results1.MetS-associated T2DM accounts for 75%(118/158) of newly diagnosed 158 T2DM cases. Among these individuals,71.4%(70/98) are male and 80%(48/60) are female.2. A total of 158 cases of T2DM patients, aged 33-80 years.<50-year-old crowd MetS prevalence was 65.7%(23/35), while the population≥50 years old MetS prevalence was 76.4%(101/123). MetS prevalence increased with age, age≥60 years old, MetS prevalence rate of 74.1%(60/81), is<60 years people MetS prevalence rate was 35.1%(37/77) of the nearly 2-fold.3. The patients with MetS-associated T2DM had higher BMI, TCHO, TG, LDL, UA, FPG, SBP, DBP, HOMA-IR and hs-CRP than did their healthy counterparts (P<0.05). When compared to patients with non-MetS-associated T2DM, patients with MetS-associated T2DM had significantly higher levels of TCHO, TG, UA, blood pressure and HOMA-IR (P<0.05) and significantly lower levels of serum folate and vitamin B12 (P<0.05). In addition, patients with MetS-associated T2DM had higher BMI and hs-CRP levels in comparison to patients with non-MetS-associated T2DM; however, this difference was not statistically significant.4. The serum leve s of UA, TG, HOMA-IR and CRP were significantly higher in T2DM patients with a TT genotype than in those with a CC or CT genotype (P<0.05 for TG vs. CRP and P<0.001 for UA vs. HOMA-IR). In contrast, the levels of vitamin B12 in the serum of patients with a TT genotype were lower than those of patients with a CC or CT genotype (P<0.01).5.The frequencies of the MTHFR homozygous TT and heterozygous CT genotypes in T2DM patients diagnosed with MetS were 19.5% and 51.7%, respectively. These values were not only significantly higher than those in the healthy subjects (7.3% and 30.9%, respectively) but were significantly higher than those in non-MetS-associated T2DM patients (10% and 32.5%, respectively). The total T frequency of the MTHFR gene significantly increased from 22.7% and 26.3% in healthy subjects and non-MS-associated T2DM patients, respectively, to 45.3% in MetS-associated T2DM patients (x2=20.665, P<0.001)Conclusion1.The MTHFR C677T gene polymorphism may contribute to insulin resistance in Han Chinese individuals with MetS.2.MTHFR gene C677T polymorphism by increasing the levels of hs-CRP,TG and decreasing the levels of vitamin B12 in the serum of subjects with a TT genotype are related to MetS. As such, this polymorphism may play an important role in the development of MetS-associated T2DM.3.The total frequency of the T genotype significantly increased from 26.3% in T2DM patients without MetS to 45.3% in those with MetS.4. The T allele increases the susceptibility of a T2DM patient to MetS. Background and ObjectiveUrinary bladder cancer is the most common tumors,80% are urothelial cancers. Many patients with urothelial bladder carcinoma have distant metastasis when diagnosed, leading to surgical removal of poor prognosis. Therefore, it is necessary to the molecular mechanism of bladder cancer in-deep study to explore new methods of early diagnosis and treatment.MicroRNA(miRNA) is a new discovery of a length of 22 nucleotide non-coding single-stranded small molecule RNA, accounting for human gene 1%, it regulates at least 30% of the total mRNA. In recent years, the relationship of miRNA with tumor are becoming a hot spot. The role of miRNAs is similar to tumor suppressor gene and oncogene function, At present, research on miRNA and bladder cancer is very few, There is still no research on bladder cancer and microRNA expression profiles in paraffin embedded tissues are reported. We combine the foundation of previous work on bladder cancer research, to investigate miRNAs profile of bladder cancer in FFPE tissue for research materials.Materials and MethodsAll subjects including 103 patients with bladder cancer and other surgical resection of the bladder tissues from the Institute of Urology of Second Hospital of Lanzhou University, archive 1998 to 2000 and between 2004-2005 were fixed in formalin and embedded in paraffin.Microarray genechip technology and PCR are used to evaluation the effectiveness and feasibility of expression analysis miRNA from FFPE. Screening biological characteristics and differential expression of miRNA of bladder cancer. Results:1 microRNA difficult to degrade so can provide effective RNA requirements to extracted miRNA microarray;2 There are 15 upregulated genes were miR-141, miR-429, miR-200a, miR-205, miR-203, miR-200c, miR-190, miR-200b, miR-210, miR-10b, miR-425, miR-34a, miR-191, miR-31, miR-10a in bladder urothelial carcinoma compared to normal bladder epithelial tissues. Other three, miRNAs -miR-487a, miR-886-5p, miR-486-5p are downregulated genes.There are no relationship between miRNA changes and pathological stages.3 Microarray chip can effectively distinguish different stages of normal bladder tissue and tumor tissue.4. MiRNA-203 of FFPE bladder cancer was significantly increased 134-fold compared with the normal, and miRNA-141 was significantly increased 373-fold compared with the normal.5. MiRNA-200 family members (miRNA-200a, miRNA-200b, miRNA-200c, miRNA-141 and miRNA-429) of bladder cancer increased compared with normal tissues, miR-141, miR-429, miR-200a, miR-200b, miR-200c were raised over the normal 337,131,21,18,10 times.Conclusions1. Paraffin embedded tissue (FFPE) can be used as the effective detection of microRNA specimens;2. The storage time (10 years; 5-years)of FFPE tissue can effectively detect the miRNA;3 Eighteen miRNAs were significantly differentially expressed between bladder cancer versus normal tissues. Overexpression of fifteen, they are miR-141, miR-429, miR-200a,miR-205,miR-203, miR-200c, miR-190, miR-200b, miR-210, miR-10b, miR-425, miR-34a, miR-191, miR-31, miR-10a. The other three miRNA are downregulated, miR-487a, miR-886-5p, miR-486-5p.4. MiRNA-203 of FFPE bladder cancer was significantly increased 134-fold compared with the normal tissue, and miRNA-141 was significantly increased 373-fold compared with the normal. Therefore speculated that miRNA-203, miRNA-141 can be used to determine in the diagnosis of bladder cancer.5. MicroRNA have no related changes with pathological staging.6. MiRNA-200 family members (miRNA-200a, miRNA-200b, miRNA-200c, miRNA-141 and miRNA-429) may involved in bladder cancer.更多还原