本文選題：瘦素 + LPS��； 參考：《山西醫(yī)科大學》2008年碩士論文

【摘要】： 目的:觀察瘦素對小鼠胸腺細胞凋亡的保護機制,探討磷酸肌醇3羥基激酶/AKT (PI3-K/AKT)和胞漿型磷脂酶A2(cPLA2)信號轉導通路是否參與瘦素保護機制,并探討瘦素抗凋亡機制,為臨床治療提供實驗依據(jù)。 方法: 1以LPS誘導小鼠胸腺細胞凋亡為模型,用MTT法檢測不同濃度瘦素對LPS誘導胸腺細胞毒性的影響,并采用Annexin V/PI雙染色法流式細胞儀檢測不同濃度瘦素對LPS誘導胸腺細胞凋亡的影響。 2 RT-PCR檢測瘦素對小鼠胸腺細胞caspase3 mRNA表達的影響,實驗分為對照組、LPS刺激組、LPS+leptin組。 3采用Annexin V/PI雙染色法流式細胞儀檢測PI3-K/AKT特異性抑制劑LY294002和cPLA2特異性抑制劑AACOCF3對小鼠胸腺細胞凋亡的影響。 4 RT-PCR檢測瘦素對小鼠胸腺細胞cPLA2 mRNA表達的影響,且cPLA2活性試劑盒檢測瘦素對cPLA2活性的影響。 結果: 1瘦素可呈劑量依賴性的減弱LPS對胸腺細胞的毒性作用,流式細胞儀檢測表明瘦素可抑制LPS誘導的細胞凋亡,其濃度為200ng/ml時有統(tǒng)計學意義(P0.05)。 2 RT-PCR結果顯示加入瘦素后caspase3 mRNA的表達下降(P0.05),說明瘦素可通過下調caspase3 mRNA表達來抑制小鼠胸腺細胞凋亡。 3流式細胞儀檢測結果顯示瘦素可以減少LPS誘導的小鼠胸腺細胞凋亡,使胸腺細胞存活率由58%上升到65% (P0.05),LY294002 (10μmol/L)可以明顯抑制瘦素的保護作用,使胸腺細胞的存活率降至60% (P0.05)。說明瘦素可依賴PI3-K/AKT途徑參與對小鼠胸腺細胞凋亡的保護。 4流式細胞儀檢測結果顯示LPS可明顯誘導胸腺細胞凋亡(P0.05),加入cPLA2特異性抑制劑AACOCF3后,可看到LPS誘導的凋亡率有所降低,與LPS刺激組相比,差異有顯著性(P0.05),說明LPS可部分依賴cPLA2途徑誘導胸腺細胞凋亡。同時RT-PCR結果顯示瘦素可上調cPLA2 mRNA的表達,cPLA2活性試劑盒顯示leptin可抑制cPLA2的活性,差異有顯著性(P0.05),說明瘦素對抗LPS誘導的小鼠胸腺細胞凋亡部分依賴cPLA2途徑。 結論:1瘦素對LPS誘導的胸腺細胞凋亡有一定的抑制作用,提示瘦素可能參與免疫調節(jié)。 2瘦素抑制LPS誘導的胸腺細胞凋亡同時依賴PI3-K/AKT和cPLA2途徑。 3 caspase3介導瘦素抗凋亡機制。
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

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本文編號：1972123