瘦素对小鼠胸腺细胞凋亡的保护机制

发布时间：2018-06-03 08:23

本文选题：瘦素 + LPS　；参考：《山西医科大学》2008年硕士论文

【摘要】： 目的:观察瘦素对小鼠胸腺细胞凋亡的保护机制,探讨磷酸肌醇3羟基激酶/AKT (PI3-K/AKT)和胞浆型磷脂酶A2(cPLA2)信号转导通路是否参与瘦素保护机制,并探讨瘦素抗凋亡机制,为临床治疗提供实验依据。方法: 1以LPS诱导小鼠胸腺细胞凋亡为模型,用MTT法检测不同浓度瘦素对LPS诱导胸腺细胞毒性的影响,并采用Annexin V/PI双染色法流式细胞仪检测不同浓度瘦素对LPS诱导胸腺细胞凋亡的影响。 2 RT-PCR检测瘦素对小鼠胸腺细胞caspase3 mRNA表达的影响,实验分为对照组、LPS刺激组、LPS+leptin组。 3采用Annexin V/PI双染色法流式细胞仪检测PI3-K/AKT特异性抑制剂LY294002和cPLA2特异性抑制剂AACOCF3对小鼠胸腺细胞凋亡的影响。 4 RT-PCR检测瘦素对小鼠胸腺细胞cPLA2 mRNA表达的影响,且cPLA2活性试剂盒检测瘦素对cPLA2活性的影响。结果: 1瘦素可呈剂量依赖性的减弱LPS对胸腺细胞的毒性作用,流式细胞仪检测表明瘦素可抑制LPS诱导的细胞凋亡,其浓度为200ng/ml时有统计学意义(P0.05)。 2 RT-PCR结果显示加入瘦素后caspase3 mRNA的表达下降(P0.05),说明瘦素可通过下调caspase3 mRNA表达来抑制小鼠胸腺细胞凋亡。 3流式细胞仪检测结果显示瘦素可以减少LPS诱导的小鼠胸腺细胞凋亡,使胸腺细胞存活率由58%上升到65% (P0.05),LY294002 (10μmol/L)可以明显抑制瘦素的保护作用,使胸腺细胞的存活率降至60% (P0.05)。说明瘦素可依赖PI3-K/AKT途径参与对小鼠胸腺细胞凋亡的保护。 4流式细胞仪检测结果显示LPS可明显诱导胸腺细胞凋亡(P0.05),加入cPLA2特异性抑制剂AACOCF3后,可看到LPS诱导的凋亡率有所降低,与LPS刺激组相比,差异有显著性(P0.05),说明LPS可部分依赖cPLA2途径诱导胸腺细胞凋亡。同时RT-PCR结果显示瘦素可上调cPLA2 mRNA的表达,cPLA2活性试剂盒显示leptin可抑制cPLA2的活性,差异有显著性(P0.05),说明瘦素对抗LPS诱导的小鼠胸腺细胞凋亡部分依赖cPLA2途径。结论:1瘦素对LPS诱导的胸腺细胞凋亡有一定的抑制作用,提示瘦素可能参与免疫调节。 2瘦素抑制LPS诱导的胸腺细胞凋亡同时依赖PI3-K/AKT和cPLA2途径。 3 caspase3介导瘦素抗凋亡机制。
[Abstract]:Aim: to observe the protective mechanism of leptin on thymocyte apoptosis in mice, and to investigate whether the signal transduction pathway of phosphoinositol 3-hydroxy-kinase / AKT PI3-K / AKT and cytosolic phospholipase A _ 2 / cPLA2 is involved in the protective mechanism of leptin, and to explore the mechanism of leptin's anti-apoptosis. To provide experimental basis for clinical treatment. Methods: 1 using LPS induced thymocyte apoptosis as a model, the effects of leptin at different concentrations on LPS induced thymocyte toxicity were detected by MTT assay. The effects of different concentrations of leptin on apoptosis of thymocytes induced by LPS were detected by Annexin V/PI double staining flow cytometry. 2 the effect of leptin on the expression of caspase3 mRNA in thymocytes of mice was detected by RT-PCR. The mice were divided into two groups: control group (LPS-stimulated group) and lipopolysaccharide leptin group. 3 the effects of PI3-K/AKT specific inhibitor LY294002 and cPLA2 specific inhibitor AACOCF3 on thymocyte apoptosis in mice were detected by Annexin V/PI double staining flow cytometry. (4) the effect of leptin on cPLA2 mRNA expression in mouse thymocytes was detected by RT-PCR, and the effect of leptin on cPLA2 activity was detected by cPLA2 activity kit. Results: 1 leptin could attenuate the toxic effect of LPS on thymocytes in a dose-dependent manner. Flow cytometry showed that leptin could inhibit apoptosis induced by LPS, and the concentration of leptin was 200ng/ml. 2 RT-PCR results showed that the expression of caspase3 mRNA decreased after the addition of leptin, suggesting that leptin could inhibit the apoptosis of thymocytes by down-regulating the expression of caspase3 mRNA. 3 the results of flow cytometry showed that leptin could reduce the apoptosis of mouse thymocytes induced by LPS, and the survival rate of thymocytes increased from 58% to 65% P0.05% LY294002 10 渭 mol / L), which inhibited the protective effect of leptin and reduced the survival rate of thymocytes to 60% P0.05. These results suggest that leptin may participate in the protection of thymocyte apoptosis by PI3-K/AKT pathway. 4 the results of flow cytometry showed that LPS could induce thymocyte apoptosis significantly (P 0.05). After adding AACOCF3, a specific inhibitor of cPLA2, the rate of apoptosis induced by LPS was decreased, compared with that in LPS group. The difference was significant (P 0.05), indicating that LPS could induce thymocyte apoptosis partly by cPLA2 pathway. At the same time, RT-PCR results showed that leptin could up-regulate the expression of cPLA2 mRNA and CPLA2 activity. The kit showed that leptin could inhibit the activity of cPLA2, and the difference was significant (P 0.05), which indicated that leptin antagonized LPS induced thymocyte apoptosis partly by cPLA2 pathway. Conclusion 1 leptin can inhibit the apoptosis of thymocytes induced by LPS, suggesting that leptin may be involved in immune regulation. 2 leptin inhibits LPS induced thymocyte apoptosis and depends on both PI3-K/AKT and cPLA2 pathways. 3 caspase3 mediated leptin antiapoptotic mechanism.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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