【作者】 袁巍；

【导师】 郑筱祥；

【作者基本信息】 浙江大学 ， 生物医学工程， 2009， 博士

【摘要】 糖尿病血管并发症是糖尿病患者致死、致残的主要原因。作为糖尿病的共同特征，高血糖是参与导致糖尿病血管内皮细胞及平滑肌细胞功能损伤的不同发病机制及发病环节的一个独立的致病因素。血管重构（vascular remodeling，VR）是血管为适应内外环境变化而发生的结构和功能的适应性改变。高血糖，胰岛素抵抗，高胰岛素血症，内皮细胞功能紊乱，非酶促糖基化，脂质过氧化和脂质浸润，血液流变学及血流动力学异常等因素，对血管壁的急慢性刺激均可引起血管重构。血管重构是糖尿病大血管及微血管病变的始动环节及病理生理基础，它在糖尿病早期血管结构和功能异常及晚期并发症的作用日益引起关注。内皮细胞与平滑肌细胞是血管壁的主要构成成分与功能单位，动脉血管壁内皮功能损伤、平滑肌细胞增殖／凋亡比例失衡、表型改变等与血管重构密切相关，因此糖尿病患者在降血糖治疗的同时，改善内皮与平滑肌细胞的功能对预防和治疗血管并发症也同样重要。本论文结合高糖诱导的内皮与平滑肌细胞损伤模型综合评价药物对血管重构的作用。细胞阻抗分析技术能非标记、实时地反映细胞的生物学状态，本研究将基于细胞阻抗分析技术的实时细胞电子传感系统(RT-CES^TM)应用于血管平滑肌细胞生长的研究，建立了高糖（25 mM）诱导的血管平滑肌细胞损伤模型和动态监测平滑肌细胞生长情况的方法。本研究实时地监测了细胞从贴附、伸展、增殖到融合，以及加入高糖刺激引起的电极阻抗的变化过程，揭示了高糖作用下平滑肌细胞生长的动态变化过程。将其与传统的WST-1细胞活性检测法进行了比较，验证了这种分析方法反映细胞数量的可靠性。此模型的建立对高血糖参与糖尿病血管损伤的发病机制研究和抗糖尿病血管并发症药物筛选评价有重要的指导意义。黄芪甲苷是中药黄芪的主要活性成分，是一种小分子量的皂苷。黄芪甲苷具有多种药理作用，包括抗糖尿病，心血管疾病及糖尿病肾病等。但是黄芪甲苷对糖尿病血管重构的影响及其作用机理还不清楚。我们观察了黄芪甲苷对高糖诱导的平滑肌细胞增殖活力及细胞凋亡的影响。结果显示，50μg／mL黄芪甲苷可显著降低平滑肌细胞的增殖活力（P＜0．01）并且使凋亡率显著增高（P＜0．01），表明黄芪甲苷可通过降低平滑肌细胞增殖活力和促使细胞凋亡来抑制平滑肌细胞的增殖。我们的结果提示抑制细胞DNA合成与抗凋亡蛋白Bcl-2表达可能是黄芪甲苷降低高糖诱导的平滑肌细胞的增殖，促进细胞凋亡的机制。另外，黄芪甲苷还可以通过促进α-SMA蛋白表达抑制平滑肌细胞去分化。PPAR-γ具有促进细胞分化，调节细胞周期等作用，我们的研究表明，黄芪甲苷具有促进平滑肌细胞PPAR-γ基因表达的作用，说明黄芪甲苷对高糖诱导的平滑肌细胞增殖的保护作用与PPAR-γ相关。为了进一步考察黄芪甲苷是否具有治疗糖尿病血管并发症的潜力，我们研究黄芪甲苷对内皮的保护作用。研究表明黄芪甲苷可改善高糖诱导的内皮屏障功能损伤，并剂量依赖性的减低TNF-α引起的内皮细胞功能损伤，包括抑制细胞代谢活性丧失和细胞凋亡，证明了它具有改善内皮功能、治疗糖尿病心血管并发症的潜力。综上，本研究得到以下新的发现与结果：（1）应用RT-CES动态地监测平滑肌细胞的生长过程，建立了高糖诱导的平滑肌细胞损伤实时监测模型，并将其与传统的WST-1细胞活性检测法进行了比较，验证了这种分析方法反映细胞数量的方法是可靠的。（2）我们的研究发现黄芪甲苷可能通过如下机制对内皮细胞及平滑肌细胞功能发挥保护作用：①通过抑制细胞周期进程降低平滑肌细胞增殖活力；②通过调节抗凋亡蛋白Bcl-2促进平滑肌细胞凋亡；③通过促进α-SMA蛋白表达调节平滑肌细胞的表型；④促进平滑肌细胞PPAR-γ基因表达；⑤改善高糖诱导的内皮屏障功能损伤；⑥抑制TNF-α引起的内皮细胞代谢活性丧失和凋亡。（3）结合高糖诱导的内皮与平滑肌细胞损伤模型综合评价了黄芪甲苷对糖尿病血管重构的作用。我们的结果表明黄芪甲苷对高糖诱导的内皮细胞及平滑肌细胞的功能损伤具有一定的保护作用，因而对糖尿病血管重构具有保护作用，在预防治疗糖尿病血管并发症方面具有一定的开发前景。更多还原

【Abstract】 Cardiovascular complications remain as the major cause of morbidity and mortalityin patients who suffer type 2 diabetes mellitus. As a common characteristic,hyperglycemia is an independent factor contributing to dysfunction of ECs andVSMCs.Vascular remodeling is a dynamic process that involves the structural alteration of avessel in response to hemodynamic and humoral stimuli. Different factors such ashyperglycemia, hyperinsulinemia, increased oxidative stress, and diabeticdyslipidemia can all contribute to alterations in the normal vessel wall andsubsequent vascular remodeling. Vascular remodeling, characterized byextracellular matrix deposition and an increased media-to-lumen ratio, occurs indiabetes and contributes to the development of complications. Vascular remodelinghas been postulated to be mediated by dysfunction of endothelial cells （ECs） and animbalance in the ratio proliferation/apoptosis of vascular smooth muscle cells（VSMCs） within the artery wall. Thus, in parallel with controlling blood glucoseconcentration, treatment of vascular complications is also important.In this study, real-time cell electrical sensing system (RT-CES^TM), which couldreflect cell biological conditions in real-time and label-free, was introduced toinvestigate the growth characteristics of VSMCs. Model of VSMC dysfunctioninduced by high glucose （25 mM） and method monitoring the VSMC growthdynamically were established as well. The whole process of electrode impedancealteration from cell adhesion, spreading, proliferation and confluence with thestimulation of high glucose was detected, indicating the dynamic changes of VSMCgrowth induced by high glucose. To ascertain that the RT-CES units of Cell Indexcorrelate with the number of the cells, comparative evaluation of impedancemeasurements and classic WST-1 assay was used. The model may be meaningful tostudy on mechanisms of diabetic vascular injury and screening of drugs for diabeticvascular complications.AstragalosideⅣ（3-O-β-D-xylopyranosyl-6-O-β-D-glucopyranosylcycloastra-genol）is a saponin extracted from radix astragali, which has been proved effective intreatment of diabetes, cardiovascular diseases and diabetic nephropathy. However, the effects of astragalosideⅣon the vascular remodeling in diabetic complicationsand the underlying mechanisms have not been well studied. We studied the effectsof astragalosideⅣon high glucose-induced alterations of proliferation andapoptosis in VSMCs. The results demonstrated that 50μg/mL astragalosideⅣreduced the viability of VSMCs enhanced the apoptotic rate of VSMCssignificantly（P＜0.01）. These results indicated that astragalosideⅣcan inhibitVSMC proliferation through reducing cell viability and promoting the apoptotic rateof VSMCs. Inhibition of the DNA synthesis and the expression of antiapoptoticprotein Bcl-2 in VSMCs may be the underlying mechanisms. In addition,astragalosideⅣmay also resist from dedifferentiation induced by high glucose inVSMCs through enhancing the expression ofα-SMA. Our data showed thatastragalosideⅣpromoted the expression of PPAR-γmRNA in VSMCs. BecausePPAR-γplay an important role in regulation of the cell cycle and differentiation, ourresults indicated that the protective effects of astragalosideⅣon high glucoseinduced proliferation in VSMCs were closely related to the altered expression ofPPAR-γ. In order to assess the therapeutic potential on diabetic vascularcomplications of astragalosideⅣ, we investigated whether astragalosideⅣhad theeffects of endothelial protection. Our results proved that astragalosideⅣcouldimprove endothelial dysfunction induced by high glucose and does-dependentinhibit TNF-α-induced cell viability loss and apoptosis. The results indicated thatthe drugs had therapeutic potential to improve endothelial dysfunction and thus thediabetic vascular complications.In short, our study achieved the following novel findings: （1）Our study usedRT-CES monitoring the growth of VSMCs dynamically and established thereal-time detecting model which could screen and assess drugs for diabeticcomplications. The model will be helpful to related drug screening, exploitation andmechanism investigation. Based on this model, we investigated the protectiveeffects of astragalosideⅣon the dysfunction of ECs and VSMCs and its relatedmechanisms. （2）Our data demonstrated that astragalosideⅣcan exert vascularprotection by inhibiting VSMCs proliferation, enhancing apoptotic rate of VSMCs,inhibiting the endothelial barrier dysfunction and inhibiting the viability loss andapoptosis of ECs, all of which contributes to the inhibitive effects of astragalosideⅣon patholygical vascular remodeling. Our study used the model of dysfunction of ECs and VSMCs to evaluate the effects of astragalosideⅣon diabetic vascularremodeling and found that astragalosideⅣhad the inhibitive effects onpatholygical vascular remodeling and may be helpful to prevent the diabeticcomplications.更多还原