本文選題：內(nèi)毒素 + 脂多糖　； 參考：《暨南大學(xué)》2007年碩士論文

【摘要】： 感染性休克是臨床常見的全身性危重病癥，死亡率極高。盡管抗生素能有效地控制G~-菌血癥，但其殺死G~-菌的同時(shí)釋放出大量內(nèi)毒素，引起內(nèi)毒素血癥，，嚴(yán)重者引起內(nèi)毒素性休克。內(nèi)毒素性休克常伴有心肌細(xì)胞損傷和心功能不全，因此防治內(nèi)毒素性心功能障礙對改善內(nèi)毒素血癥患者的預(yù)后具有重要意義。 本研究用內(nèi)毒素誘導(dǎo)的心肌細(xì)胞損傷模型，觀察甘氨酸對內(nèi)毒素所致心肌損傷的拮抗作用，為甘氨酸防治內(nèi)毒素性心肌損傷提供實(shí)驗(yàn)依據(jù)。實(shí)驗(yàn)分為三個(gè)部分進(jìn)行： 第一部分：采用MTT法觀察不同濃度Gly對LPS性心肌損傷細(xì)胞活力的影響，結(jié)果顯示藥物作用48小時(shí)后，可見隨著LPS濃度增高，Gly的保護(hù)作用隨之降低(P＜0.05)，呈一定濃度依賴關(guān)系；且Gly對照組(1.720±0.105)與正常心肌細(xì)胞(1.791±0.124)無明顯差異(P＞0.05)，提示Gly能拮抗LPS的活性，提高心肌細(xì)胞活力，效果呈濃度依賴性。 第二部分：采用Annexin V／PI流式細(xì)胞術(shù)觀察Gly對LPS性心肌損傷細(xì)胞凋亡率的影響，結(jié)果證實(shí)：Gly+LPS組的心肌細(xì)胞凋亡率均高于空白對照組(P＜0.01)；4mmol／LGly和8mmol／LGly+LPS凋亡率均低于LPS組(P＜0.01)，呈濃度依賴性；8mmol／LGly組凋亡率與空白對照組無顯著差異(P＞0.05)。提示甘氨酸能明顯抑制內(nèi)毒素所致心肌細(xì)胞凋亡，效果呈濃度依賴性。 第三部分：Gly拮抗LPS誘導(dǎo)心肌細(xì)胞凋亡的機(jī)制研究： (1)采用DIOC_6(3)染色流式細(xì)胞術(shù)觀察Gly對LPS性心肌損傷細(xì)胞線粒體膜電位的影響，結(jié)果證實(shí)：Gly+LPS組、空白對照組和Gly組的線粒體膜電位值均高于脂多糖組(P＜0.05)；同時(shí)Gly組高于空白對照組(P＜0.05)。提示Gly可以穩(wěn)定線粒體膜電位，達(dá)到對抗LPS的損傷和保護(hù)心肌細(xì)胞的效果。 (2)觀察Gly對LPS性心肌損傷細(xì)胞內(nèi)的Caspase-3活性的影響，結(jié)果證實(shí)Gly+LPS組和Gly組Caspase-3活性均低于LPS組(P＜0.05)，而Gly組與空白對照組無顯著差異(P＞0.05)。提示Gly可通過抑制Caspase-3活性，進(jìn)而抑制LPS性心肌細(xì)胞凋亡，發(fā)揮保護(hù)心肌細(xì)胞的作用。 (3)采用Western Blot法觀察Gly對LPS性心肌細(xì)胞損傷Bcl-2蛋白表達(dá)的影響，結(jié)果證實(shí)：Gly+LPS組和Gly組Bcl-2蛋白表達(dá)均高于LPS組(P＜0.05)，而Gly組與空白對照組無顯著差異(P＞0.05)。提示Gly可通過促進(jìn)Bcl-2蛋白表達(dá)，進(jìn)而抑制LPS性心肌細(xì)胞凋亡，發(fā)揮保護(hù)心肌細(xì)胞的作用。 總之，本研究結(jié)果證實(shí)：Gly能提高LPS損傷的心肌細(xì)胞的活力，抑制LPS性心肌細(xì)胞凋亡，且呈濃度依賴性；其機(jī)制與維持線粒體膜電位Δψm，抑制caspase—3的活性，提高抗凋亡蛋白Bcl-2表達(dá)，降低了LPS誘導(dǎo)的心肌細(xì)胞凋亡率有關(guān)。
[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 鹵0.105) and normal cardiomyocytes (1.791 鹵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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363

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