【作者】 庞翠军；

【导师】 周智广；

【作者基本信息】 中南大学 ， 内科学， 2007， 博士

【摘要】 研究背景:亚临床动脉粥样硬化(subclinical atherosclerosis)是动脉粥样硬化(atherosclerosis AS)的早期阶段,指患者无临床症状,但彩色B超证实颈总动脉、股动脉或髂总动脉内中膜厚度≥1.0mm和/或有动脉粥样硬化斑块出现。2型糖尿病(T2DM)多因素干预的主要目的之一是预防亚临床AS的发生发展。B类清道夫受体(scavenger receptor class B,Scarb,SRB)是LDL-c、HDL-c代谢中的重要受体蛋白,包括CD36和B类清道夫受体BⅠ(scavenger receptorclass B typeⅠ,Scarb1,SRB1)两种。CD36抗原是一种细胞膜糖蛋白,存在多种配体(如氧化的和乙酰化的LDL-c等),参与包括脂质代谢和动脉粥样硬化在内的许多生理和病理过程。SRB1是CD36蛋白家族成员,与CD36共享30%的同源序列,为脂蛋白受体中唯一能真正介导细胞与HDL-c作用的膜受体。研究目的:对新诊2型糖尿病进行4年的多因素干预,了解代谢控制状况及亚临床AS的发生情况;通过检测CD36、SRB1基因多态性、外周血单核细胞表面CD36表达情况,分析它们与T2DM患者代谢水平及亚临床AS进程的关系。第一部分新诊2型糖尿病亚临床动脉粥样硬化的前瞻性多因素干预研究目的探讨不同方案干预下新诊断2型糖尿病(T2DM)患者的代谢指标控制及亚临床动脉粥样硬化(AS)发生和发展的情况。方法采用前瞻性开放研究,将170例(35-70岁)病程1年以内、无AS的新诊T2DM患者按随机数字表法分为四组,分别为:A组(强化降糖+降压治疗)、B组(强化降糖+降压+调脂治疗)、C组(在B组基础上加服维生素E0.2g/天)及D组(在B组基础上加服复方丹参滴丸30丸/天)。共观察4年,每月随访一次,定期复查空腹血糖(FBS)、餐后2小时血糖(PBS)、糖化血红蛋白(HbAlc)、血脂、收缩压(SBP)、舒张压(DBP)、体重指数(BMI)、腰臀比(WHR)和血管彩超,了解干预4年的代谢控制情况及其颈总动脉(CCA)、股动脉(FA)内中膜厚度(IMT)和/或AS斑块发生的进展。结果①170例患者中,实际完成4年多因素干预共149例,失访21例(脱失率12.4%)。②干预4年结束时,149例患者HbAlc、甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白(LDL-c)、SBP、DBP水平均较基线显著下降(p<0.01);WHR、HbAlc、TG、TC、LDL-c、SBP、DBP达标率分别为29.5%、54.4%、74.5%、71.1%、73.8%、98.0%和98.7%,较基线显著提高(p<0.01);③共有88例发生亚临床AS,发生率达59.1%;A、B、C、D四组患者亚临床AS发生率分别为69.4%、53.8%、62.2%、51.4%,各组间比较p>0.05;④A、B、C、D四组CCA-IMT、FA-IMT均较基线增加,各组与基线比较p均<0.01;B、C、D三组干预4年后低密度脂蛋白(LDL-c)、胆固醇(TC)显著低于A组(p分别为<0.01、<0.05)。结论多因素强化干预尽管可使T2DM患者代谢状况得到很大改善,WHR、HbAlc、TG、TC、LDL-c、SBP、DBP达标率显著提高,但不能完全阻止大血管病变的发生,随着病程的增加,亚临床AS呈进展趋势。第二部分清道夫受体B(CD36、及SRB1)基因多态性与2型糖尿病及其亚临床动脉粥样硬化进程的关系目的探讨清道夫受体B基因(CD36及SRB1基因)多态性与多因素干预下T2DM亚临床AS发生的关系。方法采用PCR-RFLP方法检测470例湖南地区汉族T2DM患者及220例无糖尿病对照组CD36(CD36-rs1984112、CD36-T620C位点)及SRB1-rs5888基因多态性,比较它们基因型及等位基因频率的差异。采用logistic回归模型分析CD36-1984112及SRB1-rs5888基因型及代谢指标对亚临床AS的影响,预测多因素干预下发生亚临床AS的影响因素。结果①T2DM患者与健康受试者CD36(CD36-rs1984112位点、CD36-T620C位点)基因型及等位基因频率比较均无显著性差异(p>0.05);SRB1基因型及等位基因频率在T2DM与对照组间比较差异有显著性(p<0.01),T2DM患者携带SRB1-rs5888C/C基因型的频率低于健康人群(46.6%vs 63.2%,p<0.01),携带SRB1-C/T的频率高于健康人群(43.4%vs 30.5%,p<0.01),T2DM患者携带等位基因T的频率高于健康人群(31.7%vs 24.5%,p<0.01);②将所有携带CD36-rs1984112A/A和/或SRB1-rs5888T/T的单倍体型1、2、3、6、9合并为一类(单倍体型A),其他的单倍体型4、5、7、8合并为一类(单倍体型B),两者比较单倍体型A亚临床AS的发病率较B为高(65.4%vs 52.1%,p=0.133);③以亚临床AS是否发生(0、1)为因变量做logistic回归分析显示,多因素干预下影响亚临床AS发生的因素有3个:年龄(OR值1.103)、LDL-c(OR值2.552)、吸烟(OR值2.242),CD36与SRB1基因型及其交互作用、其他代谢指标均未进入logistic回归模型。结论①SRB1-rs5888基因型及等位基因频率在T2DM患者与健康受试者中分布不同,T2DM患者携带SRB1等位基因T的频率高于健康人群。②多因素干预下,除年龄外,LDL-c和吸烟是亚临床AS发生的主要危险因素,CD36及SRB1基因多态性可能与亚临床AS进程无关。第三部分2型糖尿病单核细胞表面清道夫受体CD36的表达及其影响因素的分析目的了解2型糖尿病患者外周血单核细胞表面CD36的表达情况,探讨影响CD36表达的有关因素及CD36表达与亚临床AS的关系。方法采用PCR-RFLP方法检测102例湖南地区汉族T2DM患者和8例健康受试者CD36-1984112及SRB1-rs5888基因多态性,采用密度梯度离心法分离T2DM患者和健康受试者外周血单个核细胞,流式细胞仪检测单核细胞表面CD36蛋白表达荧光强度,比较T2DM无AS组、T2DM亚临床AS组及健康对照组CD36的表达。多元线性回归分析CD36表达的影响因素。Logistic回归分析多因素干预下亚临床AS与CD36表达的关系。结果T2DM亚临床AS组外周血单核细胞CD36的平均荧光强度(MFI)高于T2DM无AS组(1382.23±658.69 vs 1173.02±339.71道数,p=0.047)。CD36高表达组SBP较低表达组为低,p=0.020,校正年龄后,p=0.010。多元线性回归分析显示,影响T2DM患者CD36表达的因素有:年龄(p=0.005)、性别(p=0.021)、SBP(p=0.027),标化偏回归系数分别为0.28、0.31、-0.21;男、女性T2DM患者分别做多元线性回归分析,影响男性CD36表达的因素为年龄(p=0.002);影响女性CD36表达的因素为DBP(p=0.001)。以是否存在亚临床AS(0、1)为因变量,以CD36单核细胞表面表达的荧光强度等指标作为自变量做logistic回归,留在方程的因素是年龄(p=0.004)、LDL-c(p=0.095),CD36-MFI未进入方程。结论①T2DM亚临床AS组外周血单核细胞表面CD36表达较无AS组高。②年龄、性别、SBP是CD36表达的影响因素,增龄、男性、低的SBP可使CD36表达增加;影响男性CD36表达的主要因素是年龄;影响女性CD36表达的因素主要为DBP,DBP越低,CD36的表达越高。③未发现单核细胞表面CD36的表达与亚临床AS的发生有关。更多还原

【Abstract】 Background Subclinical atherosclerosis specifies the early stage of atherosclerosis (AS),when the presence of intima-medial thickness (IMT≥1.0mm) of common carotid artery (CCA),femoral artery (FA) or common iliac artery (CIA) and/or atherosclerotic plaque was found by color ultrasound in the absence of clinical symptoms. One of the primary purposes of multi-approach intervention on Type 2 Diabetes (T2DM) is to block the progression of subclinical atherosclerosis among these patients. Scavenger receptor class B (Scarb,SR-B) is a group of important receptor protein family, including CD36 and scavenger receptor class B type I (Scarbl,SRBl). CD36 antigen is a cellular membrane glucoprotein, binding to many ligands such as oxidized and acetylizad low density lipoprotein-cholesterol (ox-LDL-c and Ac-LDL-c), participating various physiology and pathology processes, such as lipid metabolism and AS. SRB1 is one member of CD36 protein family, sharing 30% homologisation with the CD36 sequence. SRB1 is the only lipoprotein receptor on membrane that mediates the cellular binding to high density lipoprotein( HDL). Objective To investigate the progress of subclinical atherosclerosis in type 2 diabetes patients under multi-approach intervention and its association with polymorphisms of Scavenger receptor class B genes. Four-year multi-approach intervention was carried out in a group of newly diagnosed type 2 diabetes (T2DM) patients. The metabolic status and incidence of subclinical atherosclerosis were monitored periodically. Together with genotyping of CD36 (CD36-rs1984112 and CD36-T620C) and SRBl-rs5888 polymorphisms and phenotyping of CD36 expression by flow cytometry in peripheral blood monouclear cells (PBMC),the associations among CD36/SRB1 gene polymorphisms, metabolic status and the progression of subclinical atherosclerosis were analized. PartⅠThe progress of subclinical atherosclerosis in newly diagnosed type 2 diabetes patients under multi-approach intervention - a prospective clinical observationObjective To investigate the metabolic status and the incidence/progression of subclinical atherosclerosis (AS) in newly diagnosed type 2 diabetes (T2DM) patients under multi-approach intervention.Methods In this prospective case-controlled study, one hundred and seventy newly diagnosed type 2 diabetes patients without AS (35～70 years-old, duration≤1 year) were recruited and allocated into 4 groups (random digits table): group A (intensified control of blood glucose and blood pressure levels), group B (intensified control of blood glucose, blood pressure and blood lipid levels), group C (vitamin E was prescribed on the base of Group-B regimen), group D (Compound Danshen Pill was prescribed on the base of Group-B regimen). All patients were followed-up once a month in a 4-year period. Fasting blood sugar (FBS), postprandial blood sugar (PBS), HbA1c (Glycosylated hemoglobin), lipid profile, SBP (systolic blood pressure), DBP (diastolic blood pressure), BMI (body mass index), WHR (waist-to-hip ratio) and the intima-media thickness (IMT) or AS plaques of common carotid artery (CCA) and femoral artery (FA) were evaluated regularly. Results 149 of the 170 cases complied the intervention protocol in the four-year follow up (the losed rate is 12.4%), thus were taken into the final analysis. The levels of HbA1c, TG, TC, LDL-c, SBP and DBP at the end of intervention were significantly lower than the baseline (p<0.01). Also, the normalization rate of WHR, HbA1c, TG, TC, LDL-c, SBP and DBP was 29.5%、54.4%、74.5%、71.1%、73.8%、98.0% and 98.7% respectively, which was significantly higher than that at the beginning of intervention (p<0.01). Subclinical AS was found in 88 patients (59.1 %) by the end of the fourth year. The levels of CCA-IMT and FA-IMT were significantly higher than the baseline levels in group A, B, C and D (p<0.01). The incidence rate of subclinical AS was 69.4%, 53.8%, 62.2% and 51.4% in the four group, respectively (p>0.05). In group B, C and D, serum LDL-c and TC levels were significantly lower than those in group A(p<0.01,p<0.05).Conclusions Multi-approach intervention significantly improved the metabolic status of T2DM patients in terms of both levels and normalization-rate of WHR,HbA1c,TG,TC,LDL-c,SBP and DBP. However, the present intervention protocol did not stop the progression of subclinical AS in the 4-year observation. Part II The association between polymorphisms of Scavenger receptor class B genes and progress of Subclinical Atherosclerosis in Type 2 Diabetic patientsObjective To investigate the role of polymorphisms of Scavenger receptor class B genes (CD36 and SRB1 gene) in affecting the progress of Subclinical Atherosclerosis (AS) in newly diagnosed type 2 diabetics under multi-approach intervention.Methods 470 cases of T2DM and 220 non diabetic controls from Hunan, southern China were typed for CD36 (CD36-rs1984112, CD36-T620C) and SRBl-rs5888 polymorphisms using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. The genotypes and allele frequencies were compared between cases and controls. Logistic regression model was used to analysis the risk factors contributing to the progress of subclinical atherosclerosis (AS) in newly diagnosed type 2 diabetics under multi-approach intervention.Results The genotypes and allele frequencies of CD36-rsl984112 in T2DM were not significantly different between cases and controls (p>0.05), either did CD36-T620C (p>0.05). Yet the genotype and allele frequencies of SRBl-rs5888 were found significantly different between T2DM and controls (p<0.01): the frequency of SRB1-rs5888C/C genotype in T2DM was lower than that in controls (46.6% vs. 63.2%, p<0.01), the frequency of SRBl-rs5888C/T genotype in T2DM was higher than that in controls (43.4% vs. 30.5%, p<0.01), SRB1-rs5888 T allele frequency in T2DM was higher than that in controls (31.7% vs. 24.5%, p<0.01). When haplotypes carrying CD36-rs1984112 A/A and /or SRB1-rs5888T/T were combined to haplotype A, and other haplotypes were classified into haplotype B, the incidence of subclinical AS was 65.4% for haplotype A and 52.1% for haplotype B respectively (p=0.133). Logistic regression analysis revealed that the risk factors contributing to subclinical AS were age (OR = 1.103), LDL-c(OR = 2.552) and smoke (OR =2.242), while the genotypes of CD36, SRB1, and the interaction term of both were not significant factors.Conclusions SRB1-rs5888 T allele might increase risk of T2DM in Han population in southern China. Age, low-density lipoprotein cholesterol and cigarette smoking were the major determinants of the onset of subclinical AS in T2DM patients. Part III The study of CD36 expression of monocyte surface in T2DM and effecting factor of CD36 expressionObjective To investigate associated factors affecting the expression of CD36 on the surface of peripheral blood monouclear cells (PBMC) in T2DM, and the association between CD36 expression and progress of subclinical atherosclerosis.Methods 102 cases of T2DM and 8 non diabetes controls from Hunan, southern China were recruited in this study. The fluorescence intensity of CD36 on the surface of PBMC was analyzed by flow cytometry.CD36-rsl984112 and SRBl-rs5888 polymorphisms were typed by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. Multiple linear regression was used to evaluate the relevant factors contributing to CD36 expression. Logistic regression model was used to analysis the relationship between CD36 expression and subclinical atherosclerosis in type 2 diabetics under multifactorial intervention.Results The mean florescence intensity (MFI) of CD36 in T2DM with subclinical atherosclerosis was higher than that in the T2DM without atherosclerosis (1382.23±658.69 vs. 1173.02±339.71, p=0.047). SBP (systolic blood pressure) was significantly lower in subjects with high-level of CD36 expression than that in the low-expression subjects (p=0.020). Linear regression analysis showed that factor affecting the CD36 expression were: age (p=0.005), gender (p=0.021), SBP (p=0.027), standardized coefficients Beta was 0.28, 0.31 and -0.21, respectively. In man, age contributes to the CD36 expression levels in males (p=0.002), while in females DBP contributes to the CD36 expression levels (p=0.001). Logistic regression analysis revealed that age (p=0.004) and LDL-c (p=0.095) were remained in the equation while CD36-MFI wasn’t contributed to the onset of AS.Conclusions CD36 expression level was higher in T2DM with subclinical atherosclerosis in contrast with T2DM without atherosclerosis. Age, gender and SBP affect CD36 expression: CD36 expression on PBMC surface is higher in aging males with lower SBP; Age and DBP are factors affecting the CD36 expression levels in males and female, respectively. CD36 expression is not associated with the progress of subclinical atherosclerosis.更多还原