【作者】 夏珂；

【导师】 杨天伦；

【作者基本信息】 中南大学 ， 心血管内科学， 2012， 博士

【摘要】 第一章高胰岛素血症对冠心病患者血浆非对称性二甲基精氨酸水平的影响目的：2型糖尿病(T2DM)疾病早期或使用胰岛素治疗期间均有高胰岛素血症,高胰岛素血症促进动脉粥样硬化,是冠心病的独立危险因素。血管内皮功能障碍是动脉粥样硬化的始动环节,内皮来源的NO减少是其损伤的特征性表现,非对称性二甲基精氨酸(ADMA)是内源性NOS抑制剂,减少NO合成,为心血管死亡预测因子。本章研究拟探讨高胰岛素血症对人体血浆ADMA的影响,为高胰岛素血症损伤内皮提供依据。方法：来自2009年9月～2010年5月中南大学湘雅医院心内科冠心病患者及体检中心健康人群,共89人,分为三组：(1)正常对照组(Con)：30例；(2)冠心病组(CAD)：30例；(3)冠心病合并高胰岛血症组(CAD+Hins):29例。测身高、体重,计算体重指数(BMI)；测腰围、臀围,计算腰/臀(WHR)测血压。采集过夜空腹10h静脉血5mL,全自动生化分析仪测定：肝功能、总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)低密度脂蛋白胆固醇(LDL-C)、空腹血糖(FPG)、高敏C反应蛋白(hs-CRP)、尿酸(UA)、尿素氮(BUN)、血肌酐(Cr); AXSYM全自动标记免疫化学发光仪检测空腹胰岛素(Fins)、餐后2h胰岛素(2h-PIns)。结果：1. CAD+Hins组、CAD组、Con组三组间性别、年龄、血压、肝功能无显著性差异,存在可比性。2. CAD+Hins组BMI, FPG, Fins,2h-Pins, HOMA-IR均显著高于CAD组和Con组(P<0.05),CAD组上述指标显著高于Con组(P<0.05)。3.各组间TC无统计学差异,CAD+Hins组和CAD组的TG、HDL-C以及UA. Cr, BUN均显著高于Con组(P<0.05);CAD组LDL-C显著高于Con组(P<0.05); CAD+Hins组UA显著高于CAD组(P<0.05)。4. CAD+Hins组ADMA, hs-CRP水平显著高于CAD组和Con组(P<0.05)；CAD组ADMA显著高于Con组(P<0.05)。5.未发现血浆ADMA与其他生化指标的相关性。结论：1.高胰岛素血症加重已患冠心病患者的糖、脂代谢与尿酸代谢障碍。2.高胰岛素血症引起冠心病患者血浆ADMA水平进一步升高,是内皮功能损伤加重的表现；3.合并高胰岛素血症的冠心病患者血浆hs-CRP水平升高,提示高胰岛素血症与慢性炎症有关。第二章高胰岛素血症大鼠血浆ADMA/NO和主动脉SREBP、DDAH变化及与内膜损伤关系目的：第一章临床研究发现合并高胰岛素血症的冠心病患者血浆ADMA水平显著高于单纯冠心病患者与健康对照,提示高浓度胰岛素对内皮的损伤作用。二甲基精氨酸二甲胺水解酶(DDAH)是水解ADMA的关键酶,调节体内ADMA浓度。DDAH启动子区含有能与固醇调节元件结合蛋白(SREBPs)相结合的胆固醇调节组件(SRE),提示SREBPs可结合SRE调节DDAH的表达；研究表明：高胰岛素有促进SREBPs表达的作用。探讨高胰岛素血症SD大鼠主动脉SREBPs、DDAH的表达有助于进一步阐述高胰岛素损伤内皮的具体机制。本章研究拟建立高胰岛素血症SD大鼠动物模型,直接阐述高胰岛素对主动脉内膜损伤作用并探讨其可能机制。方法：采用持续皮下注射胰岛素方法建立高胰岛素血症SD大鼠模型(Hins)(N=7),并设立对照组SD大鼠(Con)(N=7)。大鼠处死前测体重,尾静脉采血测空腹血糖(FBG)；心脏穿刺法留取空腹血标本,全自动生化分析仪测总胆固醇(TC),甘油三酯(TG),高密度脂蛋白胆固醇(HDL-C)及低密度脂蛋白胆固醇(LDL-C),高敏C反应蛋白(hs-CRP); ELISA法血浆空腹胰岛素(Fins),计算胰岛素抵抗指数(HOMA-IR);比色法测血浆NO、高效液相色谱(HPLC)法测血浆ADMA浓度。大鼠处死后立刻开胸,分离升主动脉组织,快速存入液氮,4%甲醛固定。提取大鼠主动脉组织蛋白,Western-Blotting法检测主动脉SREBP-1、SREBP-2, DDAH-1、DDAH-2蛋白表达；Trizol法提取主动脉组织mRNA, RT-PCR法检测上述基因表达。结果：1.Hins组大鼠血浆空腹血糖、胰岛素、HOMA-IR与甘油三酯(TG)均显著高于Con组大鼠(P<0.05),高胰岛素血症SD大鼠模型成功。2. Hins组大鼠体重显著性高于Con组大鼠(P<0.05)。3.两组大鼠胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)无统计学差异。Hins组大鼠ADMA、hs-CRP显著高于Con组(P<0.05),NO显著低于Con组(P<0.05)。4.与Con组大鼠相比,Hins组大鼠主动脉组织SREBP-1、SREBP-2蛋白与基因表达显著升高(P<0.05),DDAH-1、DDAH-2蛋白与基因表达显著下降(P<0.05)。5.主动脉形态学改变：Con组大鼠主动脉内膜光滑,细胞排列整齐,层次清晰；Hins组大鼠主动脉内膜不规整,细胞排列紊乱。结论：1.持续皮下注射胰岛素可制作高胰岛素血症SD大鼠模型,为研究高胰岛素对机体的影响提供了可行的动物模型；2.高胰岛素血症SD大鼠血浆ADMA浓度升高、N0浓度降低,提示内皮功能受损；3.高胰岛素血症SD大鼠主动脉内膜形态发生改变,为内皮受损表现；4.高胰岛素血症SD大鼠主动脉组织SREBP-1、SREBP-2基因与蛋白表达增加,DDAH1、DDAH2基因与蛋白表达降低,可能是高胰岛素损伤血管内皮新机制。第三章高胰岛素对脐静脉内皮细胞DDAH1、DDAH2基因和蛋白表达的影响及其机制研究目的：高胰岛素血症是心血管事件的重要危险因素,我们前期临床试验和动物实验发现：高胰岛素抑制DDAH基因与蛋白表达,促进SREBPs基因与蛋白表达。在此基础上,本章通过细胞实验,进一步证实高胰岛素对内皮细胞DDAH1和DDAH2基因与蛋白表达的影响,并利用siRNA转染技术,抑制SREBP-1和SREBP-2基因表达,以探讨高胰岛素引起内皮细胞损伤的具体机制。方法：1、采用不同浓度的胰岛素处理人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells, HUVEC),并分为24小时和48小时处理组,测定葡萄糖摄取率,分析不同浓度胰岛素、作用不同时间对细胞摄取葡萄糖能力的影响。2、顺利完成siRNA转染预实验后,将HUVEC分为两大组,分别用于siRNA转染SREBP-1和SREBP-2实验,siRNA转染SREBP-1实验具体分组如下：(1)A1组：正常培养HUVEC48h;(2) B1组：培基中6×10-8mol/L胰岛素培养HUVEC细胞48h；(3)C1组：HUVEC转染siRNA-NC,6R10-8mol/L胰岛素培养48h；(4)D1组：HUVEC转染siRNA-SREBP1,正常培养48h；(5)E1组：HUVEC转染siRNA-SREBP1,6×10-8mol/L胰岛素培养48h。siRNA转染SREBP-2实验具体分组如下：(6)A2组：正常培养HUVEC48h;(7) B2组：培基中6×10-8mol/L胰岛素培养HUVEC细胞48h;(8)C2组：HUVEC转染siRNA-NC,6×10-8mol/L胰岛素培养48h；(9)D2组：HUVEC转染siRNA-SREBP2,正常培养48h；(10)E2组：HUVEC转染siRNA-SREBP2,6×10-8mol/L胰岛素培养48h。细胞经上述处理完毕后,提取各组mRNA和蛋白,Real-Time PCR检钡SREBP-1、SREBP-2、 DDAH1和DDAH2的基因表达,Western-Blot检测上述蛋白表达。结果：1.低浓度胰岛素促HUVEC摄取葡萄糖,随着胰岛素浓度的升高,HUVEC的葡萄糖摄取率逐渐增高,当胰岛素浓度超过一定范围后,随着胰岛素浓度的升高,HUVEC的葡萄糖摄取率反而随之下降。2.6×10-8mol/L胰岛素孵育HUVEC48h显著诱导HUVEC中的SREBP-1和SREBP-2的基因和蛋白表达(P<0.05),同时显著诱导转染si-RNA-NC组的SREBP-1和SREBP-2的基因和蛋白表达(P<0.05)。3.6×10-8mol/L胰岛素孵育HUVEC48h显著抑制HUVEC的DDAH1、2基因与蛋白表达(P<0.05),同时显著抑制转染si-RNA-NC组的SREBP1、2基因与蛋白的表达(P<0.05)。4.单纯转染siRNA-SREBP-1和siRNA-SREBP-2组不能上调DDAH1、2基因和蛋白表达,但转染了siRNA-SREBP-1、2的高胰岛素处理组DDAH1、2基因与蛋白的下调作用被显著削弱(P<0.05)。结论：1.高浓度胰岛素抑制人脐静脉内皮细胞摄取葡萄糖,可能与损伤有关；2.高浓度胰岛素促进人脐静脉内皮细胞SREBP-1和SREBP-2的基因和蛋白表达,抑制其DDAH1和DDAH2的基因和蛋白表达；3. siRNA沉默人脐静脉内皮细胞SREBP-1和SREBP-2基因,削弱了高胰岛素对DDAH1和DDAH2的基因和蛋白表达的抑制；4. SREBP/DDAH/ADMA途径参与高浓度胰岛素损伤人脐静脉内皮细胞。更多还原

【Abstract】 Chapter1The effect of hyperinsulinemia on plasma asymmetric dimethylarginine in patients with coronary heart diseaseObjectives:Hyperinsulinemia occurs in the early stage of type2diabetes mellitus (T2DM) and the period using insulin as therapy. Hyperinsulinemia, promoting atherosclerosis, is an independent risk factor for coronary heart disease. Since endothelial dysfunction is the initiating factor of atherosclerosis, endothelial-derived NO reduction in endothelial represents endothelial damage. Endogenous NOS inhibitor, asymmetric dimethylarginine (ADMA), decreases NO synthesis, as a predictor of cardiovascular death. The present study aims to investigate the impact of hyperinsulinemia on human plasma ADMA, and want to provide the basis for the damage of ADMA on endothelium by hyperinsulinemia.Methods:89people from September2009to May2010in Department of Cardiology, and Medical Examination Center, Xiangya Hospital, Central South University were enrolled in the present study. Cases in3different groups are as:(1) Normal Control group (Con):N=30;(2) Coronary Artery Disease group (CAD): N=30;(3) Coronary Artery Disease combined with Hyperinsulinemia group (CAD+Hins):N=29. The liver function, plasma fasting blood glucose (FBG), lipids (TC, TG, LDL-C, HDL-C), renal function (UA, BUN, Cr), high-sensitivity C-reactive protein (hs-CRP) were all checked after collecting5ml after10h overnight fasting blood samples, by automatic biochemical analyzer. Fasting insulin (Fins) and2h postprandial insulin (2h-Pins) were measured by fully automatic markers of immune chemi-luminescence analyzer. Meanwhile, the age, gender, height, weight and the waistline were recorded, the blood pressure was checked, and the body mass index (BMI) was calculated for all the subjects.Results:1. Gender, age, blood pressure, liver function have no significant difference between CAD+Hins group, CAD group, and Con group.2. The BMI of the CAD+Hins group, and FPG, Fins,2h-Pins, HOMA-1R were significantly higher than the CAD group and Con group (P<0.05). And indicators above in CAD group was significantly higher than Con group (P<0.05).3. TC has no significant difference between the groups. TG, HDL-C, and UA, Cr, BUN were significantly higher in CAD+Hins, and CAD group than that of Con group (P<0.05). LDL-C of CAD group was significantly higher than Con group (P<0.05), and UA of CAD+Hins group was significantly higher than CAD group (P<0.05).4. ADMA, hs-CRP levels in the CAD+Hins group were significantly higher than both of CAD group and Con group (P<0.05); ADMA in CAD group was significantly higher than in Con group (P<0.05).5. No correlation between plasma ADMA and other biochemical markers.Conclusions:1. Hyperinsulinemia enhanced the metabolic disorders on sugar, lipid, and uric acid metabolism in patients with coronary artery disease.2. Hyperinsulinemia further increased the plasma ADMA levels in patients with coronary artery disease, which means the aggravation of endothelial dysfunction.3. Plasma hs-CRP levels was higher in coronary artery disease patients combined with hyperinsulinemia, suggesting that hyperinsulinemia has relationship with chronic inflammatory response. Chapter2Changes of plasma ADMA/NO and aortic SREBPs, DDAH in hyperinsulinemia rats, and their relationship with aortic intimal injuryObjectives:Clinical study in Chapter1has found that plasma asymmetric dimethylarginin (ADMA) level was significantly higher in coronary artery disease patients accompany by hyperinsulinemia than simple coronary artery disease and healthy controls, suggesting that high insulin concentration might injure endothelial function. Dimethylarginine dimethylaminohydrolate (DDAH), a key enzyme regulating ADMA concentrations, contains sterol regulatory element (SRE) on it’s promoter region, which allows sterol regulatory element binding proteins (SREBPs) combine with it and regulate DDAH expression. Since high insulin concentration promotes SREBPs expression, investigation of hyperinsulinemia effect on expression of DDAH through SREBPs regulation pathway might help us clarify the mechanism of high insulin concentration on endothelial injure. This study is to set up hyperinsulinemia SD rat model firstly, and then clarify directly the injury and the possible mechanism on aortic intima by hyperinsulinemia.Methods:Establish the insulin-treated animals and control group: hyperinsulinemia SD rat model group (Hins):animals underwent twice daily sc injection of insulin (N=7), and control SD rats group (Con)(N=7). Measurement of body weight before killing the rats, tail vein blood for checking fasting blood glucose (FBG), and taking fasting blood samples by cardiac puncture. The plasma lipids (TC, TG, LDL-C, HDL-C), high-sensitivity C-reactive protein (hs-CRP) were all checked by automatic biochemical analyzer. Fasting insulin (Fins) was measured by ELISA method, and then calculated the HOMA-IR. Colorimetry method for plasma NO concentration, and HPLC method for ADMA concentration were all done. Thoracotomy immediately after the rats were sacrificed, and ascending aorta organizations was isolated and stored in liquid nitrogen rapidly,4%formaldehyde fixed meanwhile. Test the proteins expression of SREBP-1, SREBP-2, DDAH1, DDAH2by Western-Blotting and the corresponding gene expression by RT-PCR.Results:1. The FBG in Hins group, and the insulin, HOMA-IR, and triglyceride (TG) were significantly higher than Con rats (P<0.05), which suggests the successful hyperinsulinemia SD rat model.2. The body weight in Hins group was significantly higher than Con group (P<0.05).3. There is no significant difference in TC, LDL-C, and HDL-C between two groups. The ADMA and hs-CRP levels were both significantly higher than Con group (P<0.05), while the NO concentration was significantly lower than Con group (P<0.05).4. Compared with Con rats, SREBP-1and SREBP-2protein and gene expression in Hins rats aortic tissue were significantly increased, while DDAH1, DDAH2results contrasted with the results of SREBPs (P<0.05).5. Aortic morphological changes:aortic intimal looks smooth and clarity, and the cells arranged in neat rows in Con group rat. While the aortic intima looks irregular and the cells looks disorder in the Hins group rats.Conclusions:1. Hyperinsulinemia SD rat model can be created by continuous subcutaneous insulin injection, which provides a viable animal model for studying the effect of hyperinsulinemia.2. Plasma ADMA concentration increased, while NO concentration decreased in hyperinsulinemia SD rat, which indicates endothelial dysfunction.3. The aortic intima morphological changes in hyperinsulinemia SD rat due to endothelial injury.4. Proteins and genes expressions of SREBP-I and2in aortic tissue of hyperinsulinemia SD rat increased, while DDAH1and2decreased. It might be the new explanation of the mechanism on the vascular endothelial injury by hyperinsulinemia. Chapter3The affects and mechanisms of high insulin on the expressions of DDAH1, DDAH2gene and protein in HUVECObjective:Hyperinsulinemia is an important risk factor of cardiology incidents. We have found that high insulin suppresses the expressions of DDAH gene and protein from former investigations in vivo. Based on the former studies, in this chapter, we move to a forward step to investigate the affect of high concentration insulin on DDAH1and DDAH2expression in human umbilical vein endothelial cells (HUVEC). Also, we will apply the siRNA transfection technology to silent SREBP-1and SREBP-2expression, and then clarify the mechanism of endothelial dysfunction by high hyperinsulinemia.Methods:1. Using different concentrations of insulin as treatments on HUVEC. The HUVEC were divided into two groups as treated with24-hour and48-hour, the glucose uptake rate was measured, and the effect of insulin concentration, different treat time on the cellular glucose uptake ability were analyzed.2. Human umbilical vein endothelial cells were divided into two groups after successful completion of the siRNA transfection preliminary experiments:one is for siRNA-SREBP1and the other is for siRNASREBP2. The details of the groups are described as:Experiment for siRNA-SREBP-1:(1) Al:normal culture HUVEC for48h;(2) B1:HUVEC cultured in6×10-8mol/L insulin for48h;(3) Cl:siRNA-NC+Insulin group:HUVEC transfected with siRNA-NC, then cultured in6×10-8mol/L insulin for48h;(4) D1: siRNA-SREBP-1group:HUVEC transfected with siRNA-SREBP-1, cultured in normal condition for48h;(5) El:siRNA-SREBP-1+Insulin group:HUVEC transfected with siRNA-SREBP-1, cultured in6×10-8mol/L insulin for48h. And the experiment for siRNA-SREBP-2:(6) A2:normal culture HUVEC for48h;(7) B2: HUVEC cultured in6×10-8mol/L insulin for48h;(8) C2:siRNA-NC+Insulin group: HUVEC transfected with siRNA-NC, then cultured in6×10-8mol/L insulin for48h;(9) D2:siRNA-SREBP-2group:HUVEC transfected with siRNA-SREBP-2, cultured in normal condition for48h;(10) E2:siRNA-SREBP-2+Insulin group: HUVEC transfected with siRNA-SREBP-2, then cultured in6×0-8mol/L insulin for48h. The mRNA and protein of SREBP-1, SREBP-2, DDAH1and DDAH2of every group were extracted for detection of gene expression with using Real-Time PCR technology, and the protein expression of SREBP-1, SREBP-2, DDAH1and DDAH2were detected by Western-Blot.Results:1. Low concentration insulin improved the glucose uptake rate of HUVEC, and the glucose uptake rate was increased gradually with the increasing of insulin concentration. But when insulin concentration is over one point, the glucose uptake rate was declined instead.2.6×10-8mol/L insulin, with48h treatment significantly induced the expression of SREBP-1and SREBP-2genes and protein in HUVEC (P<0.05) and the group without siRNA transfection (P<0.05).3.6×10-8mol/L insulin, with48h significantly inhibit the expression of DDAH1and DDAH2gene and protein in HUVEC (P<0.05), as well as the group without siRNA transfection (P<0.05).4. Transfected with siRNA-SREBP-1and siRNA-SREBP-2 alone can not raise the expression of DDAH1and DDAH2genes and proteins. But the suppression on DDAH1and DDAH2was significantly reduced in the groups with transfection of siRNA-SREBP-1and siRNA-SREBP-2(P<0.05).Conclusions:1. High insulin concentration induces dysfunction on HUVEC, which related with injury;2. High insulin concentration induces protein and gene expression of SREBP-1and SREBP-2, while suppresses DDAH1and DDAH2;3. Silent the expression of SREBP-1and SREBP-2with siRNA reduced the suppression of high insulin concentration on DDAH1and DDAH2expression of gene and protein.4. SREBP/DDAH/ADMA pathway participates the damage effect of high insulin on HUVEC.更多还原