【作者】 李扬；

【导师】 袁莉；

【作者基本信息】 华中科技大学 ， 内科学， 2011， 博士

【摘要】 第一部分：ACE2-Ang(1-7)-Mas通路对糖尿病大鼠胰岛微循环及β细胞功能的影响目的：在2型糖尿病大鼠模型通过改变ACE2-Ang(1-7)-Mas通路活性,观察其胰岛微循环变化,及其对胰岛p细胞功能的影响。方法：长期高脂高热量饮食加小剂量STZ诱导2型糖尿病大鼠模型,颈部皮下注射Ang(1-7)或加用其受体Mas阻断剂A-779(均为300μg·kg-1·d-1)以上调或阻断ACE2-Ang(1-7)-Mas通路活性,彩色微球法测胰腺微循环血流,免疫荧光染色检测胰岛内皮细胞标志分子vWF(von Willebrand factor)及胰岛素蛋白水平表达,RT-PCR检测胰岛内皮因子vWF基因水平表达,行腹腔葡萄糖耐量试验(IPGTT)及其胰岛素释放试验(IPIRT)。结果：与正常对照组相比,2型糖尿病模型大鼠胰岛形态不规则,边界模糊,结构紊乱,微循环血流量降低54.9%(P<0.01),内皮细胞数目显著减少,甚至出现血管内皮破裂,vWF免疫荧光结果半定量分析相对浓度降低48.6%, vWFmRNA相对表达量下降17.6%(P<0.05)；空腹胰岛素降低约29.4%(P<0.05),15min(第一时相)胰岛素分泌峰值降低42.2%(P<0.01),免疫荧光染色示胰岛素相对含量明显下降(P<0.01)；空腹血糖、IPGTT 15min血糖及AUCG(葡萄糖曲线下面积)均显著升高。模型大鼠经Ang(1-7)干预后,与其空白对照组相比,胰岛内皮完整性有所恢复,微循环血流量增加0.68倍(P<0.05)；vWF蛋白相对含量与mRNA相对表达量分别增加33%和32.7%(P<0.05)；空腹胰岛素升高9.6%(P>0.05),15mmin胰岛素分泌水平增加44.3%(P<0.05),胰岛素相对含量增加23.6%；空腹血糖干预后较干预前下降(P<0.05),且其下降幅度较空白对照组增大,差异有统计学意义；IPGTT 15min血糖降低9.7%(P<0.05),AUCG降低7.4%(P<0.05)。Mas受体阻断剂A-779可以显著削弱Ang(1-7)干预带来的上述效应,降低微循环血流量,空腹胰岛素下降约4.8%(P<0.05),15min胰岛素分泌水平降低约27.9%(P<0.05),胰岛素相对含量减少,空腹血糖干预前后降低幅度减小,IPGTT基础血糖及15min血糖均升高,AUCG升高11.5%(P<0.05)。结论：在2型糖尿病模型大鼠体内行Ang(1-7)干预以上调ACE2-Ang(1-7)-Mas通路活性,可改善胰岛内皮细胞结构,增加胰腺微循环血流量,提高β细胞第—时相胰岛素分泌,并部分改善血糖水平。第二部分：ACE2-Ang(1-7)-Mas通路介导的内皮细胞效应对胰岛p细胞功能的影响目的：观察ACE2-Ang(1-7)-Mas通路活性改变对糖尿病模型和单纯肥胖模型大鼠体外分离胰岛细胞的内皮细胞功能、β细胞存活与功能的直接效应,并比较这两种模型大鼠胰岛细胞对该通路的不同反应。方法：建立糖尿病模型(DM)和单纯肥胖模型(FC)大鼠,胶原酶法分离胰岛细胞,用含10%胎牛血清及抗生素的RPMI 1640培养基进行静态培养,并进行胰岛计数及活率测定。研究Ang(1-7)的剂量-效应关系及时间-效应曲线。用最佳浓度的Ang(1-7)以最佳时间分别干预DM、FC大鼠胰岛细胞,并用Ang(1-7)受体Mas阻断剂A-779阻断ACE2-Ang(1-7)-Mas通路活性,检测其内皮舒张因子一氧化氮(NO)含量,放免法检测上清胰岛素含量,离心收集胰岛细胞行RT-PCR检测早期凋亡基因Bcl-2/Bax的表达水平,并行Hoechst染色观察其凋亡水平。结果：通过对Ang(1-7)的剂量-效应及时间-效应研究,发现Ang(1-7)10nM处理胰岛细胞6h是刺激胰岛素分泌的最佳干预剂量及时间长度。Ang(1-7)干预能提高DM大鼠和FC大鼠胰岛内皮细胞所合成与释放的NO的含量,分别为24.7%和31.7％(P<0.05)。经Ang(1-7)10nM干预6h后,DM组基础胰岛素分泌含量较其对照组上升18.1%,但是差异无统计学意义；GSIS较之升高36.5%(P<0.05)；FC组基础胰岛素分泌较其对照组降低13.2%,GSIS升高至对照组的1.58倍(P<0.01)。而以A-779 100nM阻断Mas受体的作用后,DM组和FC组基础胰岛素分泌及GSIS均显著减少。经Ang(1-7)10nM干预6h后,DM大鼠和FC大鼠胰岛细胞的Bcl-2/Bax比值均升高,分别为其对照组的2.01倍(P<0.05)和1.55倍(P<0.05),并且细胞凋亡率也呈下降趋势(P>0.05)。结论：无论是在糖尿病前期状态(FC)还是糖尿病状态(DM)下,Ang(1-7)干预均能提高胰岛内皮细胞NO合成与释放,抑制胰岛细胞凋亡。Ang(1-7)干预可以改善FC大鼠胰岛β细胞功能障碍,但其效应对DM大鼠胰岛β细胞作用有限。关键词：糖尿病模型大鼠单纯肥胖模型大鼠胰岛内皮细胞功能Ang(1-7)胰岛p细胞功能胰岛素分泌NO含量凋亡基因细胞凋亡率第三部分：ACE-AngⅡ-ATIR和ACE2-Ang(1-7)-Mas的相互平衡对胰岛细胞功能的作用及其机制研究目的：在体外离体胰岛细胞上模拟ACE-AngⅡ-AT1R和ACE2-Ang(1-7)-Mas两条通路的失衡并重建平衡,研究其内皮细胞功能改变以及对β细胞存活与功能的作用和可能机制。方法：取正常大鼠胰岛细胞进行体外培养,高糖环境诱导其凋亡状态。分别予以AngⅡ干预(1组),AngⅡ+Ang(1-7)干预(2组),AngⅡ+Ang(1-7)+A-779干预(3组)。检测其内皮舒张因子一氧化氮(NO)含量,放免法检测上清胰岛素含量,离心收集细胞行RT-PCR检测内皮细胞来源的肝细胞生长因子(HGF)、胰腺十二指肠同源盒-1(PDX-1)mRNA和早期凋亡基因Bcl-2/Bax的表达水平,并行Hoechst染色观察其凋亡水平。结果：与正常对照组相比,高糖环境可使胰岛内皮细胞所合成与释放的NO含量降低40.7%(P<0.01),降低了β细胞葡萄糖刺激的胰岛素分泌(GSIS)水平(P<0.05),胰岛细胞Bcl-2/Bax相对比值降低约50%(P<0.05),细胞凋亡率显著升高,HGF和PDX-1 mRNA的相对表达量分别降低9.5%和28.8%(P<0.05)。在此状态下,AngⅡ干预更加降低胰岛内皮细胞NO含量(P<0.05),抑制β细胞GSIS水平(P<0.05),使大鼠胰岛内皮细胞HGF mRNA相对表达量下调25.4%(P<0.05)及胰岛细胞PDX-1 mRNA相对表达量下调9.8%(P<0.05)。而上调培养液中Ang(1-7)水平后,与1组相比,2组大鼠胰岛内皮细胞合成与释放的NO含量增加至其1.56倍(P<0.05),β细胞GSIS增加约97.8%(P<0.05),胰岛细胞Bcl-2/Bax相对比值提高26%(P<0.05),HGF与PDX-1 mRNA的相对表达量升高分别为55.9%和30.7%(P<0.05)；若在培养液中再添加Ang(1-7)受体Mas拮抗剂A-779,与2组相比,则胰岛内皮细胞合成与释放的NO含量呈现出下降趋势,胰岛p细胞GSIS减少,同时Bcl-2/Bax相对比值降低,胰岛细胞早期凋亡被触发,并且HGF和PDX-1 mRNA表达均又受到抑制。结论：胰岛ACE-Angll-AT1R和ACE2-Ang(1-7)-Mas两条通路的平衡可通过介导内皮细胞效应调节β细胞功能与增殖。ACE-Angll-AT1R通路活性增加可损害内皮细胞功能,抑制p细胞功能与增殖；而上调ACE2-Ang(1-7)-Mas活性重建RAS平衡可改善内皮细胞功能及其p细胞功能障碍。内皮细胞对p细胞的作用机制可能是通过调控HGF和PDX-1的：mRNA表达,从而调节β细胞的增殖与凋亡,以及胰岛素分泌功能。更多还原

【Abstract】 PartⅠEffects of ACE2-Ang(1-7)-Mas pathway on pancreatic islet microcirculation and beta cell function in type 2 diabetic model ratsObjectives:To detect the changes of pancreatic islet microcirculation and beta cell function by upregulating the activity of ACE2-Ang(1-7)-Mas pathway in type 2 diabetic model rats in vivo.Methods:Long term high-fat diet fed plus intraperitoneal injection of a low dose of streptozotocin in Wistar rats, to create a type 2 diabetic animal model. Rats were administered by Ang(1-7) (300μg·kg-1·d-1) with or without its receptor Mas antagonist A-779 for 4 wks. Pancreatic microcirculation was detected with microspheres and microvessel morphology was evaluated by endothelial biomarker von Willebrand factor(vWF) immunofluorescence, as well as vWF mRNA expressions were detected by RT-PCR. A glucose tolerance test (GTT) was performed and plasma insulin levels were determined to detect beta-cell function., and the content of insulin were detected by immunofluorescent staining.Results:Compared with nomal control group, most islets were impaired with disarrayed architecture, and islet microvessel blood flow were decreased by 54.9%(P<0.01); endothelial cells were reduced in number and even broken in morphology, and vWF protein and mRNA expression was down-regulated(P<0.05); the basic and first-phase insulin releasing were decreased significantly; fasting blood glucose、IPGTT 15min glucose and AUCG(area under the curve of glucose)were significantly increased. After Ang(1-7) intervention, islet microvessel blood flow as well as morphology were significantly improved in the whole pancreas and intra-islet. Ang(1-7) treatment potently increased vWF gene and protein expression. Ang(1-7) administration increased the basic insulin releasing by 9.6%(P>0.05) and first-phase insulin releasing by 44.3%(P<0.05). IPGTT 15min glucose and AUCG were significantly decreased. The positive effects of Ang(1-7) above were prevented by addition of A-779.Conclusions:Chronic Ang(1-7) treatment might improve pancreatic endothelial cell morphology, enhance islet microvessel blood flow in a model of type 2 diabetic rats in vivo. It might increase first-phase insulin releasing of beta cell, and lower the blood glucose in part.PartⅡACE2-Ang(1-7)-Mas mediated pancreatic endothelial effection on beta cell functionObjectives:To investigated the effect of ACE2-Ang(1-7)-Mas on endothelial cell function as well as beta cell survival and physiological function in pancreatic islets isolated from obesity model rats and type 2 diabetic model rats in vitro. Meanwhile to compare the different reaction of pancreatic islets to Ang(1-7) administration in these two model rats.Methods:Pancreatic islets were isolated from overnight-fasted adult Wistar rats (N/FC/DM) with collagenaseⅤand hand-picked using a stereomicroscope. N, normal control; FC, long term high fat-fed rats, is an obesity rat model; DM, long term high-fat diet fed plus intraperitoneal injection of a low dose of streptozotocin, is a type 2 diabetic rat model. Islets were incubated statically in RPMI 1640 medium supplemented with 10% FBS and antibiotics, and the quantity and viability were evaluated. To investigate the dose-response and time-response of Ang(1-7). Pancreatic islets isolated from FC rats and DM rats were incubated with Ang(1-7) at the most effective concentration and for the best time length, to determine its influences on NO release, insulin secretion, and Bcl-2/Bax expressions as well as cell apoptosis rate.Results:We found that Ang(1-7) lOnM was the most effective concentration to stimulate insulin secretion and islets viability of normal rats, and islets incubated with Ang(1-7) lOnM for 6h performed best. Ang(1-7) administration resulted in a significant increase of NO content (P<0.05) compared with control group in both FC and DM rats. Upon this, after incubation with Ang(1-7) 10nM for 6h, the basic insulin secretion of islets isolated from DM rats increased by 18.1%(P>0.05), while first-phase insulin secretion increased by 36.5%(P<0.05). Whereas the basic insulin secretion of islets isolated from FC rats decreased and first-phase insulin secretion increased in contrast with control group(P<0.01). These effects of Ang(1-7) above were prevented by addition of A-779. In addition, after administration with Ang(1-7) lOnM for 6h, Bcl-2/Bax was up-regulated in islets isolated from both FC and DM rats(P<0.05).Conclusions:Ang(1-7) administration could increase NO release and suppress islet cell apoptosis in both FC and DM rat islets in vitro. Ang(1-7) intervention might improve beta cell dysfunction of FC rats, while the effect on DM rats is limited.Parts III Effects of ACE-AngⅡ-ATIR and ACE2-Ang(1-7)-Mas countbalance on pancreatic islet cells and its possible mechanismObjective:To study the functional changes of endothelial cells and the impact on pancreaticβ-cell survival and function through simulating imbalance between the ACE-AngⅡ-AT1R and ACE2-Ang (1-7)-Mas pathways and restore the balance on isolated rat pancreatic islet cells in vitro.Methods:Pancreatic islets were isolated from overnight-fasted adult normal Wistar rats and cultured statically in vitro. Islets were induced apoptosis in high glucose culture medium, and were administrated with AngII(group 1), AngⅡ+Ang(1-7)(group 2), AngⅡ+Ang(1-7)+A-779(group 3), respectively. Endothelial vasodilation factor nitric oxide (NO) content was detected, and insulin content in the supernatant was evaluated by radioimmunoassay. Islet cells were collected by centrifugation and hepatocyte growth factor(HGF), pancreatic duodenal homeobox-1(PDX-1) mRNA and early apoptotic genes Bcl-2/Bax expression were analyzed by RT-PCR, and levels of apoptosis were deteced by Hoechst staining as well.Results:High glucose decreased islet endothelial cells NO synthesis and release significantly (P<0.01), reduced glucose-stimulated insulin secretion (GSIS)(P<0.05), induced apoptosis of pancreatic islet cells and inhibited HGF and PDX-1 mRNA expression (P<0.05). Upon this state, AngⅡintervention reduced NO content of islet endothelial cells compared with control group (P< 0.05), inhibitedβ-cell GSIS(P<0.05), decreased Bcl-2/Bax ratio of islet cells(P<0.05) and down-regulated HGF and PDX-1 mRNA expressions(P<0.05). While increased Ang(1-7) content in the culture medium, NO content was increased significantly than group 1,β-cell GSIS was enhanced(P<0.05), Bcl-2/Bax ratio of islet cells was increased(P<0.05), and HGF and PDX-1 mRNA expressions were up-regulated(P<0.05)as well. If added Ang(1-7) receptor Mas antagonist A-779 in the culture medium, NO content was decreased significantly than group 2,β-cell GSIS was lowered, Bcl-2/Bax ratio was decreased, and HGF and PDX-1 mRNA expressions were suppressed, suggesting that early apoptosis of pancreatic islet cells was triggered.Conclusion:The balance of ACE-AngⅡ-AT1R and ACE2-Ang (1-7)-Mas pathways in islets could regulateβcell function and proliferation through endothelial cells effects. Increased activity of ACE-AngⅡ-AT1R pathway might impair endothelial function, inhibiteβcell function and proliferation; while up-regulating ACE2-Ang (1-7)-Mas activity to restore the balance could improve endothelial cell function and ameliorateβ-cell dysfunction. The effects of endothelial cells onβcells may be through the regulation of HGF and PDX-1 mRNA expressions to influenceβcell proliferation and apoptosis, and insulin secretion.更多还原