【作者】 徐涛；

【导师】 张杰；

【作者基本信息】 武汉大学 ， 外科学， 2018， 硕士

【摘要】 目的:探究橄榄苦苷对HK-2细胞缺糖缺氧损伤的保护作用并探讨其可能的作用机制。方法:从美国ATCC细胞库购买人肾小管上皮细胞(HK-2),到货后迅速于光学显微镜下观察形态、密度、有无污染。确定无异常后,放置在培养箱中4-6h。细胞缺氧复氧模型的制备:按照95%N2,5%CO2的通气比例调节三气培养箱,模拟体外缺氧环境来处理HK-2细胞。在缺氧24小时后,复氧不同的时间,用光学显微镜观察细胞表型以明确模型的制备条件。按照不同的处理方式将细胞分组:正常对照组(Control组)、单纯缺氧24h复氧3h组(IR组)和缺氧24h复氧3h后加不同浓度的橄榄苦苷(分别为10、50、100、200μmol/L)干预组。用CCK-8检测各组细胞的A450值,与正常组比较算出细胞的存活率,Hoechst染色检测各组细胞的凋亡情况,Western Blot法测定B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关x蛋白(Bcl-2 associated X protein,Bax)的蛋白表达量。结果:1.细胞缺氧24h,复氧不同时间后,细胞表型呈现出一定的时间依赖性。但缺氧24h复氧3h即足够模拟体外的肾缺血再灌注损伤;2.CCK-8检测结果显示,橄榄苦苷能够提高HK-2细胞在缺糖缺氧环境下的细胞存活率;3.Hoechst33258染色后显微镜下可见细胞核出现核固缩、染色质凝聚和凋亡小体形成等凋亡相关改变,并呈现出一定的浓度依赖性,这说明橄榄苦苷可减少缺糖缺氧损伤引起的细胞凋亡,降低细胞凋亡率;4.Western blot结果显示,橄榄苦苷能够增加细胞Bcl-2蛋白的表达,降低Bax蛋白的表达,差异具有统计学意义(P<0.05)。结论:橄榄苦苷对HK-2细胞的缺糖缺氧损伤具有一定的保护作用,其作用机制可能与其抑制细胞凋亡有关。更多还原

【Abstract】 Objective:To investigate the protective effect and mechanism of oleuropein on HK-2 cells injured by oxygen-glucose deprivation(OGD).Methods:The HK-2 cells was purchased from the American type culture collection.After the arrival of the cells,we observe the shape,density and whether were polluted.Then,the cells were placed in the incubator 4-6h that we insure the normal of the cells.Preparation of cell hypoxia re-oxygenation model:The three gas incubator was adjusted by the ventilation ratio of 95%N2 and 5%CO2 to simulate the anoxic environment in vitro for the treatment of HK-2 cells.After 24 hours of anoxia and different times of re-oxygenation,the cell phenotype was observed by optical microscope to clarify the preparation conditions of the model.HK-2 cells were treated with different doses of oleuropein.In each group,A450 values in cells were assayed with CCK-8(Cell Counting Kit-8)test,Afterward the apoptosis of HK-2 cells were evaluated by fluorescence microscope after Hoechst staining,The expression of Bcl-2,Bax protein and were detected by Western blot.Results:1.cells were anoxic for 24h,and after re-oxygenation at different time,the cell phenotype showed a certain time dependence.However,hypoxia 24h re-oxygenation 3h is sufficient to simulate renal ischemia-reperfusion injury in vitro.2.CCK-8 test results show:In contrast with the control group,Oleuropein could increase the survival rate of HK-2 cells.3.Apoptosis related changes were observed after Hoechst33258 staining,including nuclear condensation,chromatin condensation and apoptotic body formation and presented in a concentration dependent manner.This showed oleuropein can reduce cell apoptosis caused by OGD.4.Blotting showed that,Oleuropein could also increase the expression of Bcl-2 protein and decrease the expression of Bax protein,and the difference was statistically significant(P<0.05).Conelusion:Oleuropein could relieve the HK-2 cell injury induced by OGD and the protective effect may be related to inhibiting cell apoptosis.更多还原