【作者】 杨菊红；

【导师】 冯凭； 李京艳；

【作者基本信息】 天津医科大学 ， 内科学， 2009， 博士

【摘要】 目的：近期发现长寿蛋白SIRT1可改善肥胖大鼠的胰岛素敏感性，降低血糖，改善动脉粥样硬化。但SIRT1改善糖尿病的机制未明，其是否参与了2型糖尿病及其大血管并发症的发生发展尚缺乏深入的研究。本研究以高糖培养原代人脐静脉内皮细胞(HUVEC)为研究对象，研究SIRT1在其损伤中的作用及可能的机制。方法：0．25％胰蛋白酶灌注消化法分离HUVEC细胞。取生长良好的第2～3代细胞用于实验。实验分4个部分。第1部分：高糖对内皮细胞SIRT1表达及内皮细胞功能指标的影响。培养细胞分为正常对照组(5．5 mmol／L葡萄糖)、高糖培养组(33．3mmol／L葡萄糖)、甘露醇高渗对照组(5．5 mmol／L葡萄糖+27．8 mmol／L甘露醇)。细胞处理48小时。应用RT-PCR法检测SIRT1、E-选择素、eNOS、内皮素-1基因的表达情况，以Western印迹法检测培养细胞SIRT1的蛋白水平，以硝酸还原酶法检测培养细胞上清中NO的含量。第2部分：SIRT1激动剂白藜芦醇对高糖处理内皮细胞功能指标的影响。细胞分为正常对照组、高糖组、1μmol／L白藜芦醇预处理24小时+高糖组。高糖处理48小时。分析指标及方法同第一部分。第3部分：白藜芦醇对SIRT1基因“沉默”后高糖处理内皮细胞功能指标的影响。细胞分5组：正常对照组、高糖组、白藜芦醇预处理+高糖组、SIRT1 siRNA+白藜芦醇预处理+高糖组、Lipo 2000+白藜芦醇+高糖组(空载体组)。前3组细胞处理同第2部分。SIRT1 siRNA组细胞以Lipofectamine 2000脂质体为载体转染1 6．7 nmol／LSIRT1 siRNA入内皮细胞，以干扰内皮细胞SIRT1的表达，干扰后6小时更换为正常培养液，24小时后进行后续的白藜芦醇和高糖处理。空载体组单纯以Lipofectamine 2000脂质体处理内皮细胞，其余处理同SIRT1 siRNA组。分析指标及方法同第一部分。第4部分：白藜芦醇对高糖处理内皮细胞胰岛素抵抗的影响。培养细胞分为对照组、胰岛素处理组、高糖组+胰岛素处理组、1μmol／L白藜芦醇预处理24小时+高糖+胰岛素处理组。高糖处理48小时。后3组细胞以100 nmol／L胰岛素刺激30 min。检测上清中内皮细胞NO分泌含量。结果：(1)与正常对照相比，高糖培养48小时使内皮细胞SIRT1的mRNA及蛋白表达均降低(P＜0．05)；内皮细胞eNOS mRNA的表达及NO分泌降低(P＜0．01)；而反映内皮细胞损伤的因子如E-选择素及内皮素-1的表达却明显升高(P＜0．05)。甘露醇组与正常对照相比没有显著差异。(2)与高糖组相比，1μmol／L白藜芦醇预处理24小时明显升高高糖处理内皮细胞SIRT1的表达水平(P＜0．01)；同时内皮细胞eNOS的表达(P＜0．05)、NO分泌均明显升高(P＜0．01)；E-选择素、内皮素-1明显降低(P＜0．05)。(3)与单纯白藜芦醇处理组相比，SIRT1 siRNA组细胞SIRT1的mRNA及蛋白水平均显著降低(P＜0．05)，SIRT1蛋白表达的干扰效率为45．9％；内皮细胞的eNOS表达、NO的分泌明显降低(P＜0．05)，与高糖处理组没有差异，提示随SIRT1的表达“沉默”白藜芦醇促进eNOS表达、NO分泌的作用“消失”；内皮素-1的表达没有变化(P＞0．05)，均显著低于高糖组，提示随SIRT1的表达“沉默”白藜芦醇对内皮素-1的抑制作用不受影响；E-选择素的表达明显升高(P＜0．05)，与高糖处理组差异不明显，提示随SIRT1的表达“沉默”白藜芦醇对E-选择素的抑制作用也“消失”。空载体组与白藜芦醇处理组相比各指标无明显差异。(4)100 nmol／L胰岛素处理30 min明显刺激内皮细胞NO的分泌。高糖使内皮细胞胰岛素刺激的NO分泌降低，产生了内皮细胞胰岛素抵抗。但是白藜芦醇预处理可以明显促进高糖培养内皮细胞胰岛素刺激的NO分泌。P均＜0．01。结论(1)SIRT1通过促进eNOS表达及刺激NO分泌而具有舒张血管的作用，还通过抑制E-选择素的表达降低白细胞对内皮细胞的黏附。SIRT1的表达降低在高糖诱导的血管内皮细胞损伤中具有重要作用，SIRT1激动剂可保护内皮细胞不受高糖的影响，因而在预防和治疗糖尿病大血管病变中具有广阔的前景。(2)白藜芦醇可通过促进SIRT1的表达而具有促进高糖培养内皮细胞eNOS表达、NO分泌及降低E-选择素表达的作用。白藜芦醇还具有抑制内皮细胞内皮素-1表达的作用，且该作用与SIRT1无关。白藜芦醇还具有改善高糖环境下内皮细胞胰岛素抵抗的作用。因此，白藜芦醇在预防和治疗糖尿病大血管病变中具有广阔的前景。更多还原

【Abstract】 Objective:SIRT1 has been shown recently to lower plasma glucose levels,improve insulinsensitivity and atheroslerosis.To investigate the possible roles of SIRT1 in diabeticmacrovascular diseases,high glucose cultured primary human umbilical veinendothelial cells(HUVEC) is used.The underlying mechanisms are also investigated.Methods:Part 1,the expression of SIRT1 in high glucose culured HUVEC cells wereinvestigated,cells were divided into control group (5.5 mmol/L glucose),highglucose group (33.3 mmol/L glucose),hyperosmotic control group (27.8 mmol/Lmannitol+5.5 mmol/L glucose) The expression of SIRT1,eNOS,E-selectin,endothelin-1 mRNA were analyzed using RT-PCR;and the expression of SIRT1protein using Western blot.Supernatent of cells were collected for the analysis of thecontents of NO using nitric acid reductase.Part 2,the effects of SIRT1 activatorresveratrol on the function of high glucose culured HUVEC cells were investigated.Cells were divided into control group,high glucose group(48hours),1μmol/Lresveratrol pretreatment (24hours)group plus high glucose group.Part 3,the effects ofresveratrol on the functions of high glucose culured HUVEC after SIRT1“silence”were investigated.Cells were divided into control group,high glucose group,1μmol/L resveratrol pretreatment (24hours)group plus high glucose group,SIRT1siRNA or LipofectamineTM 2000 combined with resveratrol and high glucose group.For the latter two groups,cells were treated with 16.7 nmol/L SIRT1 siRNA (or not)in the assistance of Lipofectamine 2000 in order to interfere the expression of SIRT1.Part 4,cells were divided into control group,normal glucose group,high glucosegroup,1μmol/L resveratrol pretreatment (24 hours)group plus high glucose group.After 48 hours,cells in the latter three groups were stimulated by 100 nmol/L insulinfor 30 min.Nitric acid reductase was used to analyze the NO contents in supernatent.Results:(1) Compared with normal control cells,high glucose downregulated the levels ofSIRT1 both at the level of mRNA and protein(P＜0.05),and downregulated thelevels of eNOS mRNA (P＜0.01) and the secretion of NO (P＜0.01),butupregulated the levels of E-selectin (P＜0.05) and endothelin-1 (P＜0.05);the levels of SIRT1,eNOS,E-selectin,endothelin-1 and NO ofmannitol group did not change.(2) Compared with high glucose groups,1μmol/L resveratrol pretreatment for 24hours significantly increased the expression of SIRT1 mRNA(P＜0.01) and protein(P=0.01) of high glucose cultured HUVEC.Resveratrol also increased the level ofeNOS mRNA (P＜0.05),the secretion of NO (P＜0.01),but decreased the level ofE-selectin (P＜0.05),and the level ofendothelin-1 (P＜0.05).(3) After“knock out”the expression of SIRT1 of HUVEC,the up-regulating effectsof eNOS,NO and the down-regulating effects of E-selectin induced by resveratroldisappeared.But the down-regulating effects of resveratrol on the expression ofendothelin-1 did not change.The levels of SIRT1,eNOS,NO and endothelin-1 inLipofectamine 2000 group were not different from those in resveratrol alonetreatment group.(4) 100 nmol/L insulin treatment for 30 rains significantly stimulated the secretion ofNO in HUVEC.But high glucose impires the insulin roles on endothelial cells.Whenpretreatment endothelial cells with resveratrol significantly promote the stimulatingeffects of insulin on the secretion of NO in endothelial cells(P＜0.01).Conclusions:(1) SIRT1 is down-regulated when endothelial cells is cultured at high level ofglucose,SIRT1 activitor can improve the dilation of endothelial cell and downregulate the expression of E-selectin in endothelial cells cultured in high glucose,andfunctions as an endothelial cell protector,and therefore may have the great promise inthe prevention or therapy of diabetic macrovascular diseases.(2) Resveratrol can protect the endothelial cells from high glucose induced damageby stimulating the expression of eNOS and the secretion of NO and by inhibiting theexpression of E-selectin in SIRT1 dependent manner.Morover,resveratrol can inhibitthe expression of endothelin-1 in SIRT1 non-dependent manner in endothelial cellscultured in high glucose.Resveratrol may also have the potential to improve theinsulin resistance of endothelial cells induced by high glucose.In conclusion,resveratrol has the great prospects in the protection and therapy of diabeticmacrovascular diseases and atherosclerosis.更多还原