【作者】 于斐；

【导师】 李文杰；

【作者基本信息】 郑州大学 ， 流行病与卫生统计学， 2016， 博士

【摘要】 近年来,2型糖尿病(T2DM)在全球呈流行态势,其发病机制极其复杂。人们发现维生素D是维持正常的胰岛素分泌和糖耐量所必需的物质,在糖尿病的发生发展中担任着重要的角色。维生素D代谢通路关键基因维生素D受体基因(VDR)及其主要的25羟化酶基因(CYP2R1)对机体内维生素D的水平及其功能的发挥有着重要的调节作用,因此研究维生素D及其代谢相关基因多态性与T2DM的关系具有重要的意义。目的了解河南农村地区居民维生素D的营养状况及其影响因素;探讨VDR和CYP2R1基因与T2DM发生的关系。对象与方法1研究对象采用整群抽样法,在郑州侯寨乡、武陟县乔庙乡和洛阳新安县3个地区,共抽取2215人为调查对象,进行维生素D营养状况的流行病学调查研究;从中选取397例病例,794例对照进行病例对照研究;选取142对病例对照家系进行家系研究。2方法调查对象的一般人口学特征、生活方式、家族史、疾病史等基本资料,并进行身高、体重、腰围(WC)等体格检查;采集每个调查对象的空腹静脉血,测定空腹血糖(FPG),血脂谱,血清钙、血清磷、血清胰岛素(FIN)、血清25羟基维生素D3[25(OH)D3]等;提取全血DNA并采用TaqMan荧光探针法进行基因分型。采用Epi Data3.1软件进行双数据录入工作;定量资料描述采用`x±s或者P50(P25-P75),定性资料描述采用百分率或构成比;定量资料组间差异采用t检验、单因素方差分析、多因素协方差分析或非参数检验;定性资料组间差异采用c2检验;环境危险因素、基因变异与T2DM或25(OH)D3水平的关联分析用二分类单因素和多因素logistic回归模型分析;以上统计分析工作采用spss21.0统计分析软件完成。hardy-weinberg平衡检验采用shesis在线软件进行;单体型分析运用r软件中的haplo.stats软件包中的haplo.em、haplo.score和haplo.glm程序进行。基因和基因,基因与环境之间的交互作用与t2dm发病的关系研究采用多因子降维模型进行分析。结果1.调查地区居民维生素d的营养状况调查结果显示,所调查地区人群的25(oh)d3平均水平为27.51±22.33ng/ml,维生素d缺乏和不足者占调查总人口的77.9%,充足的个体仅占22.1%;血清25(oh)d3水平,男性人群高于女性,年龄大于55周岁低于55周岁以下的人群,具有初中及以上文化水平的高于小学及以下的人群,务工和办公室人员高于务农的人群,吸烟者高于不吸烟者,饮酒者高于不饮酒者,不吃和较少摄入咸菜者高于摄入较多者;腰臀比(whr)、腰身比(whtr),血清总胆固醇(tc)、高密度脂蛋白(hdl-c)水平异常患者体内的25(oh)d3水平低于正常人水平;t2dm,高血压患者体内的25(oh)d3水平低于正常人水平。差异均有统计学意义。多因素分析结果显示有十种指标与血清维生素d水平相关。文化水平和吸烟与25(oh)d3缺乏和不足程度呈负相关关系;年龄大于55岁,务农,咸菜摄入量较多,whr、whtr、hdl-c和tg异常与25(oh)d3缺乏和不足程度呈正相关关系。未发现vdr基因和cyp2r1基因多态性与25(oh)d3水平有显著的关联性。2.病例对照研究体重指数(bmi)、wc、whr异常、whtr异常、血脂类代谢异常、血压升高等肥胖相关指标与t2dm患病风险呈正相关关系;适度饮酒,重度体力活动,蔬果摄入较多与t2dm的发病风险呈负相关关系。vdr基因rs739837等位基因g和gg基因型在全人群、女性以及年龄大于55周岁人群中与t2dm的发病呈负相关关系;rs739837位点tt基因型携带者体内的甘油三酯水平高于gg基因型和tg基因型携带者;总胆固醇水平高于gg基因型携带者。rs2239179位点等位基因g和gg在全人群、男性和年龄大于55周岁人群和与t2dm的发病呈负相关关系。vdr单体型gcgg与t2dm的发病风险呈负相关关系。加权遗传风险评分分析结果显示,随着遗传风险评分的增加,t2dm的患病风险也随之增加。cyp2r1基因各位点的杂合子ag基因型可能是女性和年龄大于55周岁的人群发生t2dm的危险因素。交互作用分析结果显示,在t2dm的发病过程中,rs2228570,rs739837,rs2239179,rs10766197和rs10741657可能存在着的交互作用;vdr基因中rs2239179位点与蔬果摄入,rs739837位点与蔬果摄入和体力活动存在着的交互作用;cyp2r1基因中rs10766197与饮酒和蔬果摄入存在交互作用。3.病例对照家系研究指示病例组的父母、子女和所有一级亲属的糖尿病患病率均大于指示对照组,差异有统计学意义;被t2dm在调查的指示病例家系中存在家庭聚集现象;指示病例组的父母,同胞、子女、全部一级亲属的t2dm遗传度高于全家系的遗传度。指示病例家系的bmi,wc,上臂围,皮褶厚度,体脂含量等体格检查指标均高于对照家系;血清生化指标中fpg水平,fin水平,tg水平均高于指示对照家系,hdl-c水平低于指示对照家系;对照家系中的蔬果摄入高于病例家系;相对于对照家系,糖尿病家系具有明显糖尿病、高血脂、脑卒中、肥胖等家族史,另外糖尿病家系中肥胖的人数高于对照家系,差异均有统计学意义。在全家系和病例家系中vdr基因rs2189480的cc基因型可显著降低t2dm的发病风险,在对照家系中,隐性模型ca+cc是t2dm的保护因素。rs3847987和rs739837位点在对照家系中与t2dm的发病有关联。没有发现cyp2r1基因在不同家系中的分布存在显著性的差异。结论(1)机体内的维生素d水平与年龄、肥胖相关指标和脂肪代谢异常呈负相关关系;男性、饮酒者、抽烟者、文化水平较高者体内维生素d水平较高;高血压、t2dm患者体内维生素d水平较低。(2)调查地区居民血清25(oh)d3水平较低,维生素d缺乏和不足现象普遍,占总人数的77.9%,需加强关注,应制订合理措施提高当地人群维生素d的营养情况。(3)vdr基因rs739837和rs2239179位点与t2dm的发病风险相关,两个位点的g等位基因和gg基因型是t2dm的保护因素;另外rs739837变异与体内血脂代谢异常有关。(4)在T2DM的发病进程中,VDR基因和CYP2R1基因存在着交互作用;VDR基因与蔬果摄入、体力活动存在着交互作用。CYP2R1基因的rs10766197位点与饮酒和蔬果摄入存在交互作用。(5)T2DM指示病例家系的父母、子女和所有一级亲属的糖尿病患病率均大于指示对照组,具有一定的家族聚集性。指示病例家系的父母,同胞、子女、全部一级亲属的遗传度明显高于全家系的遗传度。(6)饮酒和增加蔬果摄入量对T2DM具有一定的预防保护作用;肥胖,血脂代谢异常等是T2DM的危险因素。更多还原

【Abstract】 Type 2 diabetes mellitus(T2DM) is a chronic disease with rapidly increased incidence rate in the recent year, and its pathogenesis is complicated. Many studies suppose that vitamin D plays an important role in the process of type 2 diabetes mellitus because it can promote the secretion of insulin and improve insulin resistance. VDR and CYP2R1 genes are the key genes on the metabolic pathways of vitamin D and may involve in the pathogenesis of T2 DM. ObjectiveThe purpose of this study was to explore the level of serum 25(OH)D3 and its influence factors in the rural population of Henan province, and to identify the relationship between the T2 DM susceptibility and the polymorphisms in VDR and CYP2R1 gene. Subject and Method1 SubjectA total of 2215 subjects were collected based on the family unit in Zhengzhou Houzhai town, Jiaozuo Wuzhi county and Luoyang Xin’an county. All the subjects were recruited in studying the vitamin D status in these areas. 397 T2 DM patients and 794 controls were screened for the case-control study. 142 T2 DM families and 142 control families were selected for the case-control family study.2 MethodFace-to-face survey was used to collect the basic information including the demographic characteristics, dietary and living behavior habits, family history of T2 DM, history of present illness and other medical issues.Anthropometry was conducted in the entire study population. 10 m L fasting venous blood was drawn from every participant. The biochemical indexes were tested including fasting plasma glucose(FPG), lipid profile, fasting insulin(FIN), and 25(OH)D3. After isolating the genomic DNA, the selected SNPs markers were genotyped automatically by Taqman fluorescence probe method. Epidata 3.1 software was used for data double entry and conformity test was also conducted. The categorical data were analyzed using the chi-square test. The continuous variables were compared using the student’s test, one-way analysis of variance(ANOVA), and multiple factorial analysis of variance or non-parameters tests. The OR(odds ratios) and 95% CI(confidence intervals) were examined between SNPs in gene or risk environmental factors and the risk of T2 DM or the level of 25(OH)D3 using unconditional logistic regression adjusting for covariates. All analyses were performed using SPSS software(Version21.0). Hardy–Weinberg equilibrium was tested using SHEsis online software. Haplotypes were estimated by program of Haplo.em, Haplo.score and Haplo.glm from Haplo.stats in R. Gene-gene interaction and gene-environment interaction were evaluated by Multifactor dimensionality reduction software(MDR). Results1. Serum vitamin D level of rural residents in HenanThe research results showed that the average serum 25(OH)D3 level was 27.51±22.33 ng/m L. The percentage of vitamin D deficiency and insufficiency was 77.9% and the sufficiency percentage was only 22.1%.The 25(OH)D3levelin the male was more than female;The 25(OH)D3 level of the participants aged above 55 years was higher than those aged below 55. More education received go together with higher 25(OH)D3 level. The 25(OH)D3 concentration in workers and officers was higher than in farmers. We found a positive association between serum levels of 25(OH)D3 and smoking and alcohol consumption,a negative association between serum level of 25OHD3 and salt vegetable intake. Low vitamin D levels were associated with obesity. Those with abnormal WHR, WHt R, serum TG and HDL-C have lower 25(OHD)3 level. The 25(OH)D3 level in T2 DM or hypertensive patients were significantly lower than those in control. The logistic regression analysis showed that ten factors was related to the serum level of25(OH)D3, the education and smoking were positively associated with the serum 25(OH)D3 level, while older age, working in farm, more salt intake, abnormal WHR, WHt R, TG and HDL-C were negatively associated with the serum 25(OH)D3 level.2. Case-control studyUnivariate and multivariate analysis indicated that the obesity index such as BMI, WC, WHR, WHt R were significantly positively associated with T2 DM. Abnormity of of blood lipid metabolic(high level TG and LDL-C, low level HDL-C), hypertension and salt vegetables intake were significantly positively associated with T2 DM, too. Alcohol drinking, physical activity and fresh vegetables and fruits intake were negatively associated with the development of T2 DM.For rs739837, the adjusted logistic regression analysis indicated that G allele and GG genotype were negatively associated with the development of T2 DM in the whole subject, females and those aged above 55. In addition, the TT genotype of rs739837 carriers had higher triacylglycerol than GG and TG genotype carriers, and had higher total cholesterol level than GG genotype carriers. For rs2239179, the adjusted logistic regression analysis indicated that G allele and GG genotype were negatively associated with the development of T2 DM in the whole subject, males, and those aged above 55.The haplotype GCGG of VDR was negatively associated with the development of T2 DM. The result of w-GRS showed that the individuals with higher level of GRS were more susceptible to T2 DM.For CYP2R1 gene, the results of subgroup analysis by gender and age showed that the heterozygotes AG may have a higher susceptibility to T2 DM than homozygote.The result of gene-gene interaction analysis showed that rs2228570, rs739837, rs2239179, rs10766197 and rs10741657 may have a significant interaction in the process of T2 DM. The result of gene-environment interaction analysis showed that rs2239179 and fresh vegetables intake as well as rs739837, fresh vegetables intake andphysical activity may have significant interaction in the T2 DM.3. Case-control family studyThe case families had higher prevalence of diabetes in parents, children and all first-degree relatives than the control families. T2 DM patients had some familial aggregation and a high degree of genetic tendency.The subjects in case families had higher levels of BMI, WC, arm circumference, skin fold, body fat, FIN, TG and LDL-C, but lower level of HDL-C than those in control families. The subjects in T2 DM families consumed more salty vegetable, less fresh vegetables and fruits than those in controls. The case families had stronger family history and more obese people than the control families.The results of association analysis of gene and the T2 DM in different family showed that the CC genotype of rs2189480 in VDR had lower risk of developing T2 DM than other genotypes in the whole families and case families and its recessive model CA+CC is the protective factors for T2 DM. The variants at rs3847987 and rs739837 in VDR were related to the risk of T2 DM in control families. There were no significant differences between case and control families at each site in CYP2R1 gene. Conclusion(1) The serum 25(OH)D3 level is negatively associated with age, obesity and lipid metabolic index. The 25(OH)D3 concentration is high in the male, drinkers,smokers and high education receivers. The serum 25(OH)D3 levels are significantlylow in T2 DM or hypertensive patients.(2) The prevalence of vitamin D deficiency and insufficiency in the investigationareas is high(77.9%), only 22.1% participants have sufficient vitamin D. Therefore,the rational measures should be established to improve the vitamin D status inthese areas.(3) The variants at rs739837 and rs2239179 in VDR are significantly associatedwith T2 DM, the G allele and GG genotypes of rs739837 and rs2239179 are theprotective factors for T2 DM. The variants at rs739837 are associated with theabnormity of blood lipid metabolic.(4) VDR and CYP2R1 may have a significant interaction in the process of T2 DM. The rs2239179 and fresh vegetables intake as well as rs739837, fresh vegetables intake andphysical activity may have significant interaction in the T2 DM. The rs10766197 in CYP2R1, fresh vegetables intake and alcohol drinking may have significant interaction in the T2 DM.(5) The case families have higher prevalence of diabetes in parents, children and all first-degree relatives than the control families. T2 DM patients have some familial aggregation and a high degree of genetic tendency.(6) Alcohol consumption and more vegetables or fruits intake are the protective factors for T2 DM patients, while obesity and abnormal of blood lipid metabolic are the risk factors for T2 DM.更多还原