【作者】 张冀；

【导师】 汪炳华；

【作者基本信息】 武汉大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 目的：观察同型半胱氨酸（HCY）在铜离子的介导下能否诱导血管内皮细胞凋亡并进一步探讨其可能的分子机制。 方法：体外培养人脐静脉血管内皮细胞，用不同浓度的HCY伴／不伴生理浓度Cu²⁺与内皮细胞作用；细胞计数板检测脱落细胞率；MTT法及乳酸脱氢酶（LDH）释放率的测定来衡量细胞的存活与损伤；Hoechst 33258荧光染色观察细胞核形态变化和DNA琼脂糖凝胶电泳检测细胞凋亡，并采用流式细胞术定量测定细胞凋亡率。本研究还检测了HCY对内皮细胞内脂质过氧化水平及细胞内的主要抗氧化酶—SOD、GSH-Px活性的影响。同时，还采用Western blot杂交检测p38MAPK磷酸化水平的变化。 结果：不同浓度（100～1000μmol／L）的HCY伴10μmol／L CuSO₄与内皮细胞共同孵育不同时间后，随HCY浓度的增加及作用时间的延长，细胞的（MTT）A_570nm值逐渐下降而细胞脱落量及LDH释放率逐渐上升。采用Hoechst 33258染色形态学观察发现，经1000μmol／L HCY加10μmol／LCu²⁺处理48h后，内皮细胞出现凋亡细胞典型的核固缩及核碎裂现象，DNA琼脂糖凝胶电泳出现了细胞凋亡典型的“DNA Ladder”。流式细胞仪的检测结果表明随HCY浓度增加及作用时间的延长，细胞凋亡率也相应增加。然而，不同浓度HCY单独作用于内皮细胞不同时间后，各项检测指标并无明显变化。此外，本研究还发现在铜离子存在下，随着HCY作用浓度的增加及作用时间的延长，各组细胞内抗氧化酶SOD、GSH-Px活性逐渐下降而MDA含量逐渐上升。 为了进一步研究HCY引起细胞凋亡的可能信号传导通路，本实验检测了p38MAPK在HCY联合铜离子作用下的变化情况，结果显示细胞受不同浓度的HCY作用后，p38MAPK总蛋白的表达量并不受影响，而磷酸化的p38MAPK的表达呈剂量依赖性的增加；加用p38MAPK特异性抑制剂SB203580后，可显著降低磷酸化的P38M卿K的表达量，同时可以明显抑制细胞凋亡的发生。 结论:、在生理浓度铜离子存在时，较低浓度（100一1000 p mol几）的HCY可以诱导血管内皮细胞凋亡。2、HCY在生理浓度铜离子存在下可能通过氧化应激损伤的机制而导致血管内皮细胞凋亡。3、在生理浓度铜离子存在时，HCY联合铜离子可通过激活p38MAPK通路而导致细胞凋亡。更多还原

【Abstract】 Objective: To observe whether homocysteine(HCY) plus copper could induce apoptosis in human vascular endothelial cell and explore its probable molecular mechanism.Method: Human umbilical venous endothelial cells were cultured, and were exposed to different concerntrations of HCY with/or not with CuSO4; Cell counting of detached cells were performed with a hematocytometer; the cell survival was monitored by MTT assay and the cellular injury was evaluated by LDH release; Hoechst 33258 staining was used to observe nuclear morphological changes and the apoptosis was measured by DNA agarose gel electrophoresis ;the percentage of apoptosis was quantified by flow cytometry. It was also observed that the influence of HCY on the content of cellular MDA and the changes in the activity of major antioxidant enzymes(SOD GSH-Px).Moreover, the phosphorylation of p38MAPK was detected by Western blot.Results: When endothelial cells were exposed to different concerntrations of HCY(100~100011 mol/L) plus 10 μ mol/L CuSO4 for different time, the value of cellular A570 decreased but detached cells and LDH release were increase gradually with the HCY concentration increasing and action time prolonging. By Hoechst 33258 staining used to observe nuclear morphological changes, we found that after the endothelial cells were exposed 1000 μ mol/L plus 10μ mol/L CuSO4 for 48h, the typical apoptosis phenomenon of condensed and/or fragment nuclei appeared and DNA ladder of typical apoptosis could be seen by DNA agarose gel electrophoresis. The percentage of apoptosis was increased by higher concentration of HCY and longer time. But we also observed that these detective indexes did not change when the endothelial cells were exposed to the different concentrations of HCY alone. Besides, the study indicated that when copper existing, the activities of antioxidant enzymes(SOD. GSH-Px) of these groups decreased and the content of MDA increased with the HCY concentration increasing and action timeprolonging.For studying the probable signal transmitting pathway of HCY-induced apoptosis furthermore, the changes of pSSMAPK were detected that when the endothelial cells were exposed to HCY plus copper. This experiment showed that the whole expression of p38MAPK didn’t change but the phosphorylation of pSSMAPK was enhanced in a dose-dependent manner when the cells were treated HCY and this effect of HCY on cells were antagonized by SB203580, a specific inhibitor of p38MAPK;the percentage of apoptosis was decreased when SB203580 added.Conclusions:1 -, With the physiologic concentration of copper, the lower concentration of HCY (100~1000 u mol/L) could induce apoptosis in vascular endothelial cells.2 The apoptosis in vascular endothelial cells induced by HCY plus copper was involved in the mechanism of oxidative stress-mediated injury.3, With the physiologic concentration of copper, HCY could induce apoptosis in vascular endothelial cells via activation of p38MAPK pathway.更多还原