糖基化对糖尿病合并冠状动脉粥样硬化性心脏病患者高密度脂蛋白胆固醇功能影响的研究

【作者】 唐博謇；

【导师】 严晓伟；

【作者基本信息】 中国协和医科大学 ， 临床医学， 2008， 博士

【摘要】 高密度脂蛋白（HDL）是促进胆固醇逆向转运、抗动脉粥样硬化病变中具有重要的作用的脂蛋白。除血浆水平外,HDL的组成和结构对其抗动脉粥样硬化作用也有重要意义,经物理或化学修饰后的HDL功能发生显著变化,甚至可产生具有致动脉粥样硬化作用的功能异常HDL。2型糖尿病患者常伴脂蛋白代谢异常,动脉粥样硬化发生率高。糖尿病患者血浆中的蛋白质在高糖环境下可发生非酶促糖基化修饰,但目前对糖基化修饰与糖尿病患者HDL功能改变之间的确切关系尚存在较大意见分歧。了解糖尿病患者HDL的糖基化修饰水平及其对HDL结构功能的影响,将有助于进一步了解糖尿病患者动脉粥样硬化性疾病的发病机制,为采取针对性的预防与诊治提供理论依据。本研究获取了糖尿病患者HDL及体外糖基化修饰的健康人HDL组分,比较了不同程度糖基化HDL的抗LDL氧化能力及诱导胆固醇外流功能;旨在明确糖基化修饰与糖尿病HDL抗氧化能力及诱导胆固醇外流功能改变间的关系,并同时考查药物等因素对其的影响。目的:建立检测HDL糖基化水平的适宜方法,探索糖尿病合并冠状动脉粥样硬化性心脏病患者HDL的糖基化水平,明确糖基化修饰与糖尿病患者HDL诱导胆固醇外流功能及抗低密度脂蛋白（LDL）氧化保护功能改变之间的关系,探索糖尿病冠心病患者细胞内胆固醇积聚的机制,并考察使用他汀类药物对糖基化修饰及HDL功能的影响。方法:1.收集冠心病合并糖尿病患者、单纯冠心病患者血浆样本共19例,健康受试者血浆样本11例;2.通过密度梯度离心方法分离高密度脂蛋白;3.健康受试者HDL在体外用不同浓度葡萄糖孵育液进行体外糖基化修饰;4.分别通过果糖胺法和三硝基苯磺酸（TNBS）法检测患者HDL的糖基化水平和体外修饰的HDL糖基化水平,并评价两种方法;以糖基化结果中位数分为高糖基化水平与低糖基化水平组进行后续功能相关性研究;5.用放射性同位素方法检测HDL在体外诱导人脐静脉内皮细胞（HUEVC）和HepG2细胞内胆固醇外流的外流率,分析外流率与HDL糖基化水平的相关性;6.将各组HDL与健康受试者低密度脂蛋白（LDL）共同孵育,加入Cu²⁺氧化LDL,通过检测紫外吸收OD234nm测定脂质氧化程度,评估HDL抗氧化能力评估,分析HDL的抗氧化能力与HDL糖基化水平的相关性;7.分析使用他汀类药物对糖尿病患者HDL胆固醇外流功能和抗LDL氧化功能的影响。结果:1.本研究中获得的HDL浓度及糖基化水平未达到果糖胺法检测灵敏度范围;2.经TNBS法检测,糖尿病患者HDL的糖基化水平高于非糖尿病患者（1.0172对0.8067,p=0.017）,但HDL的糖基化水平与HbA1c（p=0.364）及空腹血糖值（p=0.388）不相关;3.经体外糖基化修饰的HDL诱导HUEVC和HepG2细胞内胆固醇外流的外流率与糖基化水平均不相关;4.糖尿病及冠心病患者中,HDL高水平糖基化组与低水平糖基化组的胆固醇外流率间无显著性差异（11.38%对12.09%,p=0.313）;5.未经他汀类药物治疗的糖尿病患者HDL抗LDL氧化的迟滞时间(T_lag)显著低于非糖尿病患者（95.03min对102.55min,p=0.020）;6.在全部患者中,HDL的糖基化水平与其抗LDL氧化的T_lag呈负相关性（Pearson相关系数-0.504,p=0.028）,HDL高水平糖基化患者的T_lag也显著低于低糖基化水平组（90.37min对102.46min,p=0.022）;7.与各组HDL共同孵育的LDL氧化时吸光度最大值(OD_max)间无显著性差异;8.他汀类药物不影响糖尿病患者HDL的糖基化水平（0.9823对1.0730,p=0.395）,但可显著提高患者HDL诱导HUEVC内胆固醇流出的外流率（12.23%对10.28%,p=0.024）,并延长HDL抗LDL氧化的T_lag（100.68min对95.03min,p=0.020）。结论:1) TNBS法可有效检测超速离心法获得的人HDL的糖基化水平。果糖胺法的灵敏度低,不适用于检测超速离心法获得的HDL样品的糖基化水平。2)与非糖尿病患者相比,糖尿病患者HDL的糖基化水平显著升高。糖尿病患者HbA1c值或空腹血糖水平与HDL的糖基化水平不平行。使用他汀类药物不影响糖尿病患者HDL的糖基化水平。3)糖尿病患者HDL诱导胆固醇外流功能降低,使用他汀类药物可显著改善糖尿病患者HDL的外流功能。但糖基化修饰不是糖尿病患者HDL外流功能下降的直接原因。4)糖基化修饰可能是糖尿病患者HDL抗LDL氧化保护能力降低的原因之一。他汀类药物对HDL抗氧化功能的改善不依赖于其糖基化水平。更多还原

【Abstract】 High density lipoprotein（HDL） promotes reverse cholesterol transport（RCT）,thus plays important roles in many antiatherogenic pathways.In addition to serum levels, the component and structure of HDL significantly affect its functions.Physically or chemically modified HDL brought forward a series of function changing,can even turn into proatherogenic proteins.Dyslipidemia is a common finding in type 2 diabetes patients,along with increased risk of artherosclerosis.Serum proteins of patients with type 2 diabetes can carry on nonenzymatic glycation,but controversial opinions still exist on whether the functional defects of HDL correlated with it glycation levels.Understanding the distribution of glycation levels of HDL,as well as its impact on HDLs’ functions,may contributes to the treatment and prevention of artheriosclerosis in type 2 diabetes.We extracted HDL from diabetic patients’ serum samples,as well as from healthy subjects’ samples for in vitro glycation,and compared HDL mediated intracellular cholesterol efflux and anti-oxidative function between groups.Our aim was to identify the relationships between HDL glycation and its anti-oxidative function or efflux capacity by different means,as well as to examine the impact of statins on HDL glycation and function.OBJECTIVE:To discuss the mechanism of intracellular accumulation of cholesterol in diabetic patients with or without coronary artery disease（CAD）,by evaluating the effects of glycation of HDL on its efflux capacity and anti-oxidative function,as well as to establish a proper method for determination of glycation of HDL,and explore the role of statins on functions of HDL associated with glycation and diabetes.METHOD:1.Serum was collected from 19 CAD patients,with or without type 2 diabetes,as well as 11 healthy age-matched subjects;2.HDL was isolated by ultracentrifugation,from serum of both healthy subjects’ and patients’ samples;3.Mixed HDL from healthy subjects was incubated with glucose of different levels, before further experiment with patient samples;4.Glycation levels of HDL was evaluated by measuring fructosamine or trinitrophene sulphonic acid（TNBS） method.The applicability of each method was evaluated; 5.HDL mediated of cholesterol efflux from human umbilical vein epithelial cells（HUVECs） or HepG2 cells mediated by each HDL sample was measured with ¹⁴C-cholesteryl oleate labeled LDL in vitro,and the correlation with glycation level was analyzed;6.Anti-oxidative fuction was evaluated by detecting the optical density（OD,234nm） and calculating the lag time(Tl_ag) of oxidation of the LDL incubated with HDL samples,with oxidative stress provided by Cu²⁺.Correlations between anti-oxidative functions and glycation levels were analyzed;7.Effects of statins on glycation level and functions of HDL were analyzed.RESULT:1.The concentration and glycation levels of HDL in our study went beyond the sensitivity of the measurement with fructosamine method;2.According to the result of TNBS testing,the level of HDL glycation in diabetic patients was significantly higher than that in non-diabetic ones（1.0172 vs.0.8067, p=0.017）.HDL glycation levels were not correlated with HbA1c or free blood glucose levels（p=0.364 and 0.388,respectively）;3.The rates of efflux mediated by HDL glycated in vitro in HUEVC or HepG2 cells were not correlated with glycation levels of HDL;4.Among diabetic and/or CAD patients,no significant differences were observed between efflux rates among groups with high HDL glycation levels and low levels（11.38%vs.12.09%,p=0.313）;5.The Lag time of LDL oxidation in the presence of HDL was correlated HDL glycation levels（Pearson’s Correlation Coefficient -0.504,p=0.022）,and T_lag was significantly lower in the group with higher HDL glycation levels（90.37min vs. 102.46min,p=0.022）;6.OD_max of LDL oxidation were not significantly differed between groups with higher and lower HDL glycation levels;7.The use of statins didn’t affect the level of HDL glycation（0.9823 vs.1.0730, p=0.395）,however,it significantly increased cholesterol efflux in HUVECs mediated by HDL from diabetic patients（12.23%vs.10.28%,p=0.024）,as well as prolonged the lag time of LDL oxidation in the presence of HDL from patients（100.68min vs. 95.03min,p=0.020）.CONCLUSION: 1.The TNBS method can sufficiently detect glycation levels of HDL isolated by ultracentrifuge.The fructosamine method has a lower sensitivity,and is therefore,not capable for glycation level determination of ultracentrifugally isolated HDL;2.In compare with non-diabetic subjects,diabetic patients have HDL with higher glycation level.The HbA1c and free blood glucose levels can not sufficiently predict HDL glycation levels in diabetic patients.The use of statins doesn’t lower HDL glycation level;3.HDLs from diabetic patients have defected ability in inducing cholesterol efflux, which can be reverted by the use of statins.Glycation doesn’t directly impair the cholesterol efflux capacity of HDL.4.Glycation may be one of the factors causing defect anti-oxidative functions of HDLs in diabetic patients.The improvement of HDL’s anti-oxidative functions by statins is independent to the state of HDL glycation.更多还原