大鼠肺微血管内皮细胞衰老机制的研究

【作者】 吉瑞瑞；

【导师】 陈兰英； 孙仁宇；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2003， 博士

【摘要】 肺功能不全是多种肺疾病发展至最后可能产生的共有的病理过程，并由此引发多器官功能衰竭，危及生命。人口老龄化是21世纪全球面临的重大课题。提示研究包括老年肺功能不全在内的老年性疾病发生机制的迫切性。肺微血管内皮细胞(pulmonary microvascular endothelial cells，PMVECs)在肺功能不全的发病学中起主导作用。本研究拟通过研究衰老PMVECs存活与凋亡信号通路的变化规律，阐明老化过程中肺功能不全的细胞分子机制。将对老年肺功能不全以及老年相关疾病的研究提供有益探索，具有极其重要的社会意义和经济意义。 目的 在成功分离培养大鼠PMVECs的基础上，建立稳定可靠的D-半乳糖(D-galactose，D-gal)诱导的大鼠PMVECs衰老模型；从细胞周期，细胞生长存活和细胞凋亡及其信号传导途径调控等方面探讨PMVECs衰老的细胞分子机制。 方法 1．大鼠PMVECs的分离培养与鉴定。联合应用胰蛋白酶和胶原酶消化肺边缘组织分离细胞，血清诱导内皮细胞聚集。根据内皮细胞典型的铺路石样的形态学特征，运用胰蛋白酶／EDTA局部消化内皮细胞克隆再进行扩大培养。用下述方法鉴定大鼠PMVECs：(1)免疫荧光标记法检测内皮细胞内vWF；(2)荧光显微镜观察PMVECs吞噬红色荧光染料DiI标记的乙酰化的低密度脂蛋白(DiI-Ac-LDL)的功能；(3)透射显微镜观察大鼠PMVECs的超微结构 (4)根据内皮细胞能吞噬DiI-Ac-LDL的功能，运用流式细胞仪计量有荧光标记的阳性细胞，以鉴定所分离大鼠PMVECs的纯度。 2．D-gal诱导大鼠PMVECs衰老模型的建立。运用四甲基偶氮唑盐微量酶反应比色法(MTT法)观察D-gal对大鼠PMVECs生存活力的影响；将原代分离的大鼠PMVECs传代，以10g／L D-gal的DMEM培养液培养细胞，传代3次继续培养7d后终止培养，即为用于本文实验的D-gal诱导的大鼠PMVECs衰老模型。以不含D-gal的上述培养液培养传代3次的细胞为对照组。此外，将PMVECs经D-gal诱更多还原

【Abstract】 Aged pulmonary insufficiency is the possible common pathological process of many lung diseases , which can lead to multiple organ dysfunction syndrome(MODS),eventually death. It is urgent to study on molecular and cellular mechanisms of age-related diseases, including aged pulmonary insufficiency. Pulmonary microvascular endothelial cells( PMVECs )is the key point in pathogenesis of pulmonary insufficiency because of the special functions. We hope to clarify the celluar and molecular mechanisms of pulmonary insufficiency during ageing through research on the change rules by signal pathway in growth, survival and apoptosis . The subject not only will provide useful clues for the study of aged pulmonary insufficiency and age-related diseases but also has extremely important social and economic meaning..ObjectTo establish a stable and relialble model of PMVECs ageing induced by D-gal based on isolating and culturing PMVECs from rats lung successfully; Explore cellular and molecular mechanisms of PMVECs senescence on cell cycle, growth and survival ,apoptosis ,signal pathways.Methods1. Isolation, culture, purification and identification of PMVECs from rats lung. The purified PMVECs were obtained after isolated from peripheral lung tissues , digested with trypsin and collagenase and aggregated by serum stimulation. According to typical cobble morphology of endothelial cells, PMVECs were selected and transferred by trypsin/EDTA local digestion to amplify the number of cells. The following methods were used to identify PMVECs from rats lung.(l) von更多还原