甘氨酸保护心肌细胞防止内毒素性损伤的作用和机制研究

发布时间：2018-06-28 09:29

本文选题：内毒素 + 脂多糖　；参考：《暨南大学》2007年硕士论文

【摘要】： 感染性休克是临床常见的全身性危重病症，死亡率极高。尽管抗生素能有效地控制G~-菌血症，但其杀死G~-菌的同时释放出大量内毒素，引起内毒素血症，，严重者引起内毒素性休克。内毒素性休克常伴有心肌细胞损伤和心功能不全，因此防治内毒素性心功能障碍对改善内毒素血症患者的预后具有重要意义。本研究用内毒素诱导的心肌细胞损伤模型，观察甘氨酸对内毒素所致心肌损伤的拮抗作用，为甘氨酸防治内毒素性心肌损伤提供实验依据。实验分为三个部分进行：第一部分：采用MTT法观察不同浓度Gly对LPS性心肌损伤细胞活力的影响，结果显示药物作用48小时后，可见随着LPS浓度增高，Gly的保护作用随之降低(P＜0.05)，呈一定浓度依赖关系；且Gly对照组(1.720±0.105)与正常心肌细胞(1.791±0.124)无明显差异(P＞0.05)，提示Gly能拮抗LPS的活性，提高心肌细胞活力，效果呈浓度依赖性。第二部分：采用Annexin V／PI流式细胞术观察Gly对LPS性心肌损伤细胞凋亡率的影响，结果证实：Gly+LPS组的心肌细胞凋亡率均高于空白对照组(P＜0.01)；4mmol／LGly和8mmol／LGly+LPS凋亡率均低于LPS组(P＜0.01)，呈浓度依赖性；8mmol／LGly组凋亡率与空白对照组无显著差异(P＞0.05)。提示甘氨酸能明显抑制内毒素所致心肌细胞凋亡，效果呈浓度依赖性。第三部分：Gly拮抗LPS诱导心肌细胞凋亡的机制研究： (1)采用DIOC_6(3)染色流式细胞术观察Gly对LPS性心肌损伤细胞线粒体膜电位的影响，结果证实：Gly+LPS组、空白对照组和Gly组的线粒体膜电位值均高于脂多糖组(P＜0.05)；同时Gly组高于空白对照组(P＜0.05)。提示Gly可以稳定线粒体膜电位，达到对抗LPS的损伤和保护心肌细胞的效果。 (2)观察Gly对LPS性心肌损伤细胞内的Caspase-3活性的影响，结果证实Gly+LPS组和Gly组Caspase-3活性均低于LPS组(P＜0.05)，而Gly组与空白对照组无显著差异(P＞0.05)。提示Gly可通过抑制Caspase-3活性，进而抑制LPS性心肌细胞凋亡，发挥保护心肌细胞的作用。 (3)采用Western Blot法观察Gly对LPS性心肌细胞损伤Bcl-2蛋白表达的影响，结果证实：Gly+LPS组和Gly组Bcl-2蛋白表达均高于LPS组(P＜0.05)，而Gly组与空白对照组无显著差异(P＞0.05)。提示Gly可通过促进Bcl-2蛋白表达，进而抑制LPS性心肌细胞凋亡，发挥保护心肌细胞的作用。总之，本研究结果证实：Gly能提高LPS损伤的心肌细胞的活力，抑制LPS性心肌细胞凋亡，且呈浓度依赖性；其机制与维持线粒体膜电位Δψm，抑制caspase—3的活性，提高抗凋亡蛋白Bcl-2表达，降低了LPS诱导的心肌细胞凋亡率有关。
[Abstract]:Although antibiotics can effectively control Gluta-bacteremia, it can release a large amount of endotoxin while killing Gnomonas, causing endotoxemia and endotoxin-induced shock in severe cases. Endotoxic shock is often associated with myocardial cell injury and cardiac insufficiency, so prevention and treatment of endotoxin-induced cardiac dysfunction is of great significance in improving the prognosis of patients with endotoxemia. In this study, the antagonistic effect of glycine on endotoxin-induced myocardial injury was observed in order to provide experimental evidence for the prevention and treatment of endotoxin-induced myocardial injury by glycine. The experiment was divided into three parts: in the first part, MTT assay was used to observe the effects of different concentrations of gly on the viability of LPS-induced myocardial injury cells. The protective effect of gly decreased with the increase of LPS concentration (P < 0. 05), and there was no significant difference between gly control group (1.720 卤0.105) and normal cardiomyocytes (1.791 卤0.124), suggesting that gly could antagonize the activity of LPS and increase the activity of cardiomyocytes. The effect was concentration-dependent. The second part: the effect of gly on the apoptosis rate of LPS-induced myocardial injury was observed by Annexin V / Pi flow cytometry. The results showed that the apoptotic rate of myocardial cells in the control group was higher than that in the control group (P < 0. 01). Part three: Gly antagonizes the mechanism of LPS-induced cardiomyocyte apoptosis: (1) the effect of gly on the mitochondrial membrane potential of LPS-induced myocardial injury was observed by flow cytometry with DIOC6 (3) staining. These results suggest that gly can stabilize mitochondrial membrane potential and protect myocardial cells from LPS injury. (2) to observe the effect of gly on Caspase-3 activity in LPS-induced myocardial injury cells. The results showed that the activity of Caspase-3 in gly LPS group and gly group was lower than that in LPS group (P < 0.05), but there was no significant difference between gly group and blank control group (P > 0.05). These results suggest that gly can inhibit the apoptosis of LPS-induced cardiomyocytes by inhibiting the activity of Caspase-3. (3) Western blot was used to observe the effect of gly on the expression of Bcl-2 protein in LPS-induced myocardial injury. The results showed that the expression of Bcl-2 protein was higher in the Gly LPS group and the gly group than in the LPS group (P < 0.05), but there was no significant difference between the gly group and the blank control group (P > 0.05). These results suggest that gly can protect cardiomyocytes by promoting the expression of Bcl-2 protein and then inhibiting the apoptosis of LPS-induced cardiomyocytes. In conclusion, the results of this study demonstrated that the activity of caspase-3 could be increased and the apoptosis of LPS-induced cardiomyocytes could be inhibited in a concentration-dependent manner, and its mechanism was related to maintaining mitochondrial membrane potential 螖蠄 m and inhibiting the activity of caspase-3. Increasing the expression of anti-apoptotic protein Bcl-2 and decreasing the rate of apoptosis induced by LPS were related to the expression of anti-apoptotic protein Bcl-2.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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