【作者】 杜弢；

【导师】 周智广；

【作者基本信息】 中南大学 ， 内科学， 2008， 博士

【摘要】 第一部分维生素D受体基因Fok I多态性与自身免疫性糖尿病的关系目的:①探讨维生素D受体（VDR）基因Fok I多态性与自身免疫性糖尿病（T1DM和LADA）的关系;②探讨VDR基因Fok I多态性对谷氨酸脱羧酶抗体（GADA）滴度的影响。对象与方法:研究对象为T1DM患者241例,LADA患者354例,T2DM患者473例,正常对照380例。采用PCR-RFLP方法检测VDR基因Fok I多态性,放射配体法检测GADA滴度,放射免疫法检测胰岛素和C肽;分析VDR基因Fok I多态性与糖尿病、GADA滴度及胰岛β细胞功能的关系。结果:①全体观察对象VDR基因Fok I基因型符合Hardy-Weinberg平衡;②VOR基因Fok I多态性在对照组和糖尿病组间比较,糖尿病组ff基因型和f等位基因频率（22.3%,48.1%）高于正常对照（17.9%,42.8%）,而FF基因型和F等位基因频率（26.1%,51.9%）低于正常对照组（32.4%,57.2%）（P＜0.05）;③LADA组ff基因型分布频率（27.1%）高于正常对照组（17.9%）,而T2DM组FF基因型分布频率（24.1%）低于正常对照组（32.4%）,差异具有统计学意义（P值分别为0.028、0.011）;此外,T1DM组VDR基因Fok I基因型分布与LADA组基因型分布差异具有统计学意义（P=0.041）,而与对照组比较无统计学差异;携带ff基因型人群中LADA患病相对危险性增加（OR值为1.707,95%的可信区间为1.201～2.427）;④在LADA患者中,GADA滴度在ff组最高（0.1742）,Ff基因型组次之（0.1363）,FF基因型最低（0.1104）,基因型ff组显著高于FF组（P＜0.05）;⑤分别以GADA滴度0.3和0.8为切点,将LADA组分为高滴度组（GADA≥0.3,0.8,LADA-1组）和低滴度组（GADA＜0.3,0.8,LADA-2组）,无论在切点0.3或者0.8,LADA-1组与正常对照比较,VDR基因Fok I基因型分布差异均具有统计学意义（P＜0.01）,而LADA-2的Fok I基因型分布与对照组比较无差异（P＞0.10）。在切点0.8,LADA-1组与LADA-2组比较,VDR基因Fok I基因型分布差异具有统计学意义（P=0.009）;而在切点0.3,VDR基因Fok I基因型在LADA-1组与LADA-2组中的分布差异无统计学意义（P=0.179）。⑥在T1DM组,VDR基因Fok I3种基因型之间临床及代谢指标差异均无统计学意义（P＞0.05）;Ff基因型组与ff基因型组的FCP、PCP均低于FF基因型组,差异具有统计学意义。⑦在LADA组,VDR基因Fok I 3种基因型之间临床及代谢指标无差异（P＞0.05）;Ff基因型组与ff基因型组的FCP低于FF基因型组,差异具有统计学意义（P＜0.05）。HOMA-IS在FF组最高,Ff基因型组次之,ff基因型最低;HOMA-IR在ff组最高,Ff基因型组次之,FF基因型最低,但二者均无统计学意义（P＞0.05）。结论:VDR基因Fok I多态性与T1DM遗传易感性不相关,与GADA滴度亦不相关。VDR基因Fok I ff基因型与LADA风险增加有关,可能是LADA遗传易感基因之一。LADA患者VDR基因Fok I多态性与GADA滴度相关,其VDR基因Fok I ff基因型与高GADA滴度呈正相关。第二部分自身免疫性糖尿病单核细胞表面CD14、TLR2和TLR4的表达目的:观察自身免疫性糖尿病患者外周单核细胞表面CD14、TLR2和TLR4的表达。对象与方法:1型糖尿病患者（T1DM）16例、成人隐匿性自身免疫糖尿病患者（LADA）18例、2型糖尿病患者（T2DM）22例和正常对照23例,用化学分离法获得外周血单个核细胞（PBMC）后,磁珠分离法获取纯化的单核细胞,使用流式细胞仪（FCM）检测单核细胞表面CD14、TLR2和TLR4的表达。用酶联免疫法（ELISA）检测血清IL-1β和TNF-α水平。结果:LADA患者单核细胞表面CD14表达显著高于T1DM、T2DM和正常对照人群（P＜0.001）,而T1DM患者单核细胞CD14的表达较正常人群低（P=0.002）,T2DM患者单核细胞CD14水平与正常对照组相比无显著差异（P＞0.05）。LADA和T2DM患者单核细胞表面TLR4表达高于T1DM和正常对照（P＜0.05）,LADA与T2DM比较无差异（P＞0.05）。各组人群单核细胞TLR2表达均未发现有差异性（P＞0.05）。各组糖尿病患者IL-1β和TNF-α水平均较高,正常人群IL-1β低于可检测值。结论:T1DM和LADA单核细胞表面CD14、TLR2和TLR4表达的变化,提示自身免疫性糖尿病可能存在天然免疫调控的改变。第三部分维生素D3对自身免疫性糖尿病单核细胞的免疫调节作用目的:探讨1,25（OH）₂D3对自身免疫性糖尿病单核细胞的免疫调节作用。方法:体外培养的外周单核细胞分别从23例正常对照、16例1型糖尿病患者（T1DM）、18例成人隐匿性自身免疫糖尿病患者（LADA）以及22例2型糖尿病患者（T2DM）中获得。观察1,25（OH）₂D3预干预后,单核细胞对TLR2配体（脂磷壁酸,LTA）和TLR4配体（脂多糖,LPS）的反应性变化以及单核细胞表面CD14,TLR2和TLR4表达变化。流式细胞仪分析单核细胞CD14,TLR2和TLR4表达以及NF-κB-p65磷酸化水平,IL-1β和TNF-α水平用酶联免疫吸附法（ELISA）检测。结果:LPS和LTA降低T1DM,T2DM和正常对照组单核细胞表面CD14的表达,而增加LADA组单核细胞表面CD14的表达（P＜0.05）;LTA对单核细胞表面TLR2表达的影响在T1DM、LADA、T2DM和正常对照组间比较无差异（P＞0.05）;LPS使正常对照组单核细胞表面TLR4表达下降的幅度明显高于T1DM、LADA和T2DM组（P＜0.05）。LPS和LTA干预后,T1DM、LADA和T2DM单核细胞NF-κB-p65磷酸化水平和IL-1β和TNF-α浓度均明显高于正常对照组（P＜0.001）;1,25（OH）₂D3预干预后,LPS和LTA对单核细胞表面CD14、TLR2和TLR4表达、NF-κB-p65磷酸化水平和IL-1β和TNF-α浓度的影响各组间比较无差异（P＞0.05）。结论:LPS和LTA刺激可降低正常对照组和T1DM单核细胞表面CD14的表达,而升高LADA单核细胞表面CD14的表达,这可能是LADA的一个特异性表现。1,25（OH）₂D3预干预后,T1DM和LADA单核细胞在TLR配体刺激下CD14、TLR2和TLR4的表达、NF-κB-p65磷酸化水平以及IL-1β和TNF-α浓度均与正常对照组相似。研究结果表明1,25（OH）₂D3对T1DM和LADA患者单核细胞的高反应性具有一定的免疫调节作用,这有助于我们近一步地认识和探讨1,25（OH）₂D3作为自身免疫性糖尿病治疗的价值。更多还原

【Abstract】 Part one Association between the VDR gene Fok I polymorphism and autoimmune diabetesObject:To study the association between the VDR gene Fok I polymorphism and autoimmune diabetes.Subject and Methods:595 autoimmune diabetic patients including 241 typical type 1 diabetic patients and 354 latent autoimmune diabetes in adults（LADA）,473 type 2 diabetic patients and 380 unrelated healthy subjects were recruited for this study from China.Genotyping for VDR gene Fok I polymorphism were performed by RFLP-PCR technique. Multiple linear regression was used to study for associations between autoimmune antibodies levels of GADA and IA-2A and VDR gene Fok I genotype in T1DM and LADA.Results:1) The distribution of VDR gene Fok I genotypes of all subjects was in compliance with the Hardy-Weinberg equilibrium （P=0.776）.2) The distribution of VDR gene Fok I genotype and allele frequencies between diabetic patients and controls differed significantly （P＜0.05） with the ff genotype occurring more frequently in diabetic patients.3) Moreover,there was a significant difference in frequencies of VDR gene Fok I genotype and allele between T1DM and LADA （P=0.041） whereas this difference was not observed between T1DM and controls.4) VDR gene Fok I ff genotype（odds ratio[OR]=1.707,95% CI 1.201～2.427） was the main susceptibility genotype in LADA.5) Multiple linear regression showed no association of the autoimmune antibodies,IA-2A and GADA with VDR Fok I genotype in T1DM. However,in LADA ff genotype was associated with higher GADA titer compared with FF genotype（P＜0.05）.6) LADA patients were divided into LADA-1 and LADA-2 according to the titers higher than 0.8 or not. The distribution of VDR gene Fok I genotype between LADA-1 and LADA-2 differed significantly（P=0.009）.The distribution of VDR gene Fok I genotype in LADA-2 was no difference compared with that in controls.7) In T1DM,Ff and ff genotypes showed lower levels of FCP and PCP than FF genotype.Moreover,in LADA,Ff and ff genotypes showed lower level of FCP than FF genotype.Although low HOMA-IS and high HOMA-IR in LADA ff genotype,the difference was not significant compared with that in FF genotype（P=0.242,P=0.790; respectively）.Conclusions:1) VDR gene Fok I genotype showed no association with T1DM.2) VDR gene Fok I ff genotype may be a genetic mark for predicting risk of LADA and was associated with higher GADA titer. Part two Monocyte surface CD14,TLR2 and TLR4 expression in autoimmune diabetesObject:To investigate the CD14,TLR2 and TLR4 expression of monocyte from T1DM,LADA and T2DM.Methods:Peripheral blood monocytes were collected from 23 healthy controls,16 type 1 diabetes mellitus（T1DM）,18 latent autoimmune diabetes in adults（LADA）,and 22 type 2 diabetes mellitus （T2DM）,respectively.CD14,TLR2 and TLR4 expression were analyzed by flow cytometer.Serum IL-1βand TNF-αlevel were measured by enzyme linked immunosorbent assay（ELISA）.Results:Monocytes showed significantly higher surface CD14 expression from LADA compared with that from T1DM,T2DM and controls（P＜0.001）.However,surface CD14 expression on monocytes from T1DM was lower than controls（P=0.002）.Monocyte surface CD14 expression from T2DM showed no difference compared with controls （P＞0.05）.Higher TLR4 expression on freshly isolated monocytes from LADA and T2DM compared that from T1DM and controls（P＜0.05）.And no difference of monocyte TLR4 expressin between LADA and T2DM was found（P＞0.05）.The levels of TLR2 expression were no significant differences between patients and controls（P＞0.05）.Levels of IL-1βand TNF-α,as inflammation biomarkers,was high in patients,concentration of IL-1βin controls was under-detected.Conclusions:Changes of monocyte surface CD14,TLR2 and TLR4 expressions in T1DM and LADA suggests that the change of the innate immune may be involved in autoimmune diabetes. Part three Modulation of 1,25-dihydroxy-vitamin D3 on monocyte hyperresponsiveness from autoimmune diabetesObject:To investigate modulation of 1,25-dihydroxy-vitamin D3 on monocyte activity from autoimmune diabetes.Methods:Peripheral blood monocytes were collected from 23 healthy controls,16 type 1 diabetes mellitus（T1DM）,18 latent autoimmune diabetes in adults（LADA）,and 22 type 2 diabetes mellitus （T2DM）,respectively.The effect of 1,25-dihydroxy-vitamin D3 （1,25（OH）₂D3） on monocyte response to TLR2 ligand（lipoteichoic acid, LTA） and TLR4 ligand（lipopolysaccharide,LPS） was evaluated in vitro by measuring phosphorylation level of NF-κB-p65 and associated cytokine production（IL-1βand TNF-α）.In additional,effects of preincubation with 1,25（OH）₂D3 on moncyte CD14,TLR2 and TLR4 expression stimulated by LTA and LPS were observed.CD14,TLR2 and TLR4 expression and phosphorylation level of NF-κB-p65 were determined by flow cytometer（FCM）.IL-1βand TNF-αconcentrations were measured by enzyme-linked immunosorbent assay（ELISA）.Results:LPS and LTA decreased surface expressions of CD14 were observed on monocytes from T1DM,T2DM and controls,in contrast to increased on monocytes from LADA（P＜0.05）.After incubation with LPS,the levels of monocyte TLR4 expression remarkably decreased in controls,as compared with T1DM and LADA（P＜0.05）.There was no significant difference in the expression of TLR2 on monocyte between patients and controls after stimulation with LTA（P＞0.05）.Additionally, expressions of CD14,TLR2 and TLR4 on monocytes surface did not show significant changes between patients and controls when monocytes were pretreated with 1,25（OH）₂D3,and afterwards incubated with LPS or LTA（P＞0.05）.Activation of NF-κB and amounts of IL-1βand TNF-αproduction by stimulation with LTA and LPS significantly increased in T1DM and LADA,which was modulated by 1,25（OH）₂D3 to similar level,as compared to controls.Conclusions:Monocytes from T1DM and LADA showed similar high cellular reactivity towards ligands and 1,25（OH）₂D3 was observed to restore this defect to a certain extent in vitro.The modulation of 1,25（OH）₂D3 on monocytes makes us to consider more potency of vitamin D3 as therapy in autoimmune diabetes.更多还原