DHA对脂肪细胞凋亡调控作用机制研究

发布时间：2018-05-28 16:41

本文选题：二十二碳六烯酸 + 3T3-L1脂肪细胞　；参考：《宁波大学》2011年硕士论文

【摘要】：目的以3T3-L1脂肪细胞为模型探讨DHA对前体脂肪细胞和成熟脂肪细胞凋亡的影响及其机制,为应用DHA进行肥胖的防治提供科学依据。方法3T3-L1前脂肪细胞常规培养,MTT法检测DHA处理24、48、72h对3T3-L1前脂肪细胞活力的影响,流式细胞术检测DHA作用24h对前脂肪细胞周期的影响,Hoechst染色法观察DHA处理24h对前脂肪细胞凋亡的影响,线粒体膜电位JC-1染色法检测DHA处理24h前脂肪细胞线粒体膜电位的改变,蛋白免疫印迹法检测DHA处理对前脂肪细胞中p-ERK1/2、p21、Bcl-2、Bax和p53蛋白水平的影响,试剂盒法检测DHA处理后前脂肪细胞凋亡效应因子Caspase 3的活性变化,从而探讨DHA对前脂肪细胞凋亡的影响及其作用机制。MDI法诱导前体脂肪细胞分化为成熟脂肪细胞并进行以下检测:MTT法检测DHA处理对成熟脂肪细胞活力的影响,油红O染色法测定DHA处理48h对成熟脂肪细胞脂质含量的影响,Hoechst染色法观察DHA处理48h对成熟脂肪细胞凋亡的影响,JC-1染色法检测DHA处理24h成熟脂肪细胞线粒体膜电位的改变,蛋白免疫印迹法检测DHA处理对成熟脂肪细胞凋亡相关蛋白Akt、p-Akt、CREB、ERK1/2、p-ERK1/2、Bcl-2、Bax和Bad水平的影响,试剂盒法检测DHA处理48h对成熟脂肪细胞凋亡效应因子Caspase 3活性的影响,从而研究DHA对成熟脂肪细胞凋亡的影响及其作用机制。结果MTT结果表明DHA可降低3T3-L1前脂肪细胞的活力。对3T3-L1前脂肪细胞周期的研究发现,DHA能诱导前脂肪细胞G2/M期阻滞,增强ERK1/2的活化、上调p21的表达。Hoechst染色显示DHA可诱导3T3-L1前脂肪细胞凋亡,JC-1染色表明DHA可降低线粒体膜电位,蛋白免疫印迹分析发现DHA可上调凋亡相关蛋白p53表达、降低Bcl-2/Bax比值。同时发现DHA可增强前脂肪细胞凋亡效应因子Caspase 3的活性。对成熟脂肪细胞的研究发现,DHA可降低3T3-L1成熟脂肪细胞的活力、降低脂质含量,Hoechst染色结果显示DHA能诱导3T3-L1成熟脂肪细胞凋亡,JC-1染色表明DHA处理24h可降低成熟脂肪细胞的线粒体膜电位,蛋白免疫印迹分析发现DHA可降低Bcl-2/Bax比值、抑制成熟脂肪细胞抗凋亡蛋白Akt和ERK1/2的活化、抑制CREB表达,上调促凋亡蛋白Bad水平,还发现DHA可增强成熟脂肪细胞凋亡效应因子Caspase 3的活性。结论DHA可激活ERK1/2/p21途径诱导G2/M期阻滞,抑制3T3-L1前脂肪细胞增殖,并通过上调p53表达、降低Bcl-2/Bax比值、升高Caspase 3活性,诱导前脂肪细胞凋亡。DHA通过抑制Akt/CREB和ERK途径、降低Bcl-2/Bax比值、上调促凋亡蛋白Bad水平、升高Caspase 3活性,诱导成熟脂肪细胞凋亡。
[Abstract]:Objective to investigate the effect of DHA on apoptosis of preadipocytes and mature adipocytes using 3T3-L1 adipocytes as a model and to provide scientific basis for the prevention and treatment of obesity by DHA. Methods routine culture of 3T3-L1 preadipocytes was used to detect the effect of DHA treatment on the viability of 3T3-L1 preadipocytes for 72 hours. Flow cytometry was used to detect the effect of DHA on the cell cycle of preadipocytes. The effect of DHA treatment on the apoptosis of preadipocytes was observed by Hoechst staining. Mitochondrial membrane potential (JC-1) staining was used to detect the changes of mitochondrial membrane potential (MMP) in adipocytes before 24 hours of DHA treatment. Western blot was used to detect the effects of DHA treatment on the levels of p-ERK1 / 2p21 Bcl-2Bcl-2BX and p53 protein in preadipocytes. The activity of apoptosis effector factor Caspase 3 in adipocytes was detected by kit method after DHA treatment. To investigate the effect of DHA on the apoptosis of preadipocytes and its mechanism. MDI induced precursor adipocytes to differentiate into mature adipocytes. The effects of DHA treatment on the viability of mature adipocytes were detected by the following method. Effects of DHA treatment for 48 h on lipid content of mature adipocytes the effect of DHA treatment on apoptosis of mature adipocytes was observed by oil red O staining. The mitochondrial membrane potential of mature adipocytes treated with DHA for 24 h was detected by JC-1 staining. Western blot was used to detect the effect of DHA treatment on the levels of Bad and Bcl-2Bax in mature adipocyte apoptosis related protein Akttnp-AkttCor CREBERK1 / 2pERK1 / 2, and the effect of DHA treatment on the activity of Caspase 3 in mature adipocytes for 48 h. To study the effect of DHA on the apoptosis of mature adipocytes and its mechanism. Results MTT results showed that DHA could reduce the activity of 3T3-L1 preadipocytes. The study of 3T3-L1 preadipocyte cycle showed that DHA could induce G2 / M phase arrest of preadipocytes, enhance the activation of ERK1/2, and up-regulate the expression of p21. Hoechst staining showed that DHA could induce apoptosis of 3T3-L1 preadipocytes and JC-1 staining showed that DHA could decrease mitochondrial membrane potential. Western blot analysis showed that DHA could up-regulate the expression of apoptosis-related protein p53 and decrease the ratio of Bcl-2/Bax. At the same time, it was found that DHA could enhance the activity of preadipocyte apoptosis effector Caspase 3. Studies on mature adipocytes showed that DHA could reduce the activity of 3T3-L1 mature adipocytes. The results of Hoechst staining showed that DHA could induce apoptosis of 3T3-L1 mature adipocytes. DHA treatment for 24 h could reduce mitochondrial membrane potential of mature adipocytes. Western blot analysis showed that DHA could decrease Bcl-2/Bax ratio. Inhibiting the activation of antiapoptotic protein Akt and ERK1/2, inhibiting the expression of CREB and up-regulating the Bad level of apoptotic protein in mature adipocytes, it was also found that DHA could enhance the activity of Caspase 3, the apoptosis effector of mature adipocytes. Conclusion DHA can activate ERK1/2/p21 pathway to induce G _ 2 / M phase arrest and inhibit the proliferation of 3T3-L1 preadipocytes. By upregulating p53 expression, decreasing Bcl-2/Bax ratio and increasing Caspase _ 3 activity, DHA can induce preadipocyte apoptosis and decrease Bcl-2/Bax ratio by inhibiting Akt/CREB and ERK pathway. The level of apoptotic protein Bad was up-regulated, the activity of Caspase 3 was increased, and the apoptosis of mature adipocytes was induced.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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