本文選題：二十二碳六烯酸 + 3T3-L1脂肪細(xì)胞　； 參考：《寧波大學(xué)》2011年碩士論文

【摘要】：目的以3T3-L1脂肪細(xì)胞為模型探討DHA對前體脂肪細(xì)胞和成熟脂肪細(xì)胞凋亡的影響及其機(jī)制,為應(yīng)用DHA進(jìn)行肥胖的防治提供科學(xué)依據(jù)。 方法3T3-L1前脂肪細(xì)胞常規(guī)培養(yǎng),MTT法檢測DHA處理24、48、72h對3T3-L1前脂肪細(xì)胞活力的影響,流式細(xì)胞術(shù)檢測DHA作用24h對前脂肪細(xì)胞周期的影響,Hoechst染色法觀察DHA處理24h對前脂肪細(xì)胞凋亡的影響,線粒體膜電位JC-1染色法檢測DHA處理24h前脂肪細(xì)胞線粒體膜電位的改變,蛋白免疫印跡法檢測DHA處理對前脂肪細(xì)胞中p-ERK1/2、p21、Bcl-2、Bax和p53蛋白水平的影響,試劑盒法檢測DHA處理后前脂肪細(xì)胞凋亡效應(yīng)因子Caspase 3的活性變化,從而探討DHA對前脂肪細(xì)胞凋亡的影響及其作用機(jī)制。MDI法誘導(dǎo)前體脂肪細(xì)胞分化為成熟脂肪細(xì)胞并進(jìn)行以下檢測:MTT法檢測DHA處理對成熟脂肪細(xì)胞活力的影響,油紅O染色法測定DHA處理48h對成熟脂肪細(xì)胞脂質(zhì)含量的影響,Hoechst染色法觀察DHA處理48h對成熟脂肪細(xì)胞凋亡的影響,JC-1染色法檢測DHA處理24h成熟脂肪細(xì)胞線粒體膜電位的改變,蛋白免疫印跡法檢測DHA處理對成熟脂肪細(xì)胞凋亡相關(guān)蛋白Akt、p-Akt、CREB、ERK1/2、p-ERK1/2、Bcl-2、Bax和Bad水平的影響,試劑盒法檢測DHA處理48h對成熟脂肪細(xì)胞凋亡效應(yīng)因子Caspase 3活性的影響,從而研究DHA對成熟脂肪細(xì)胞凋亡的影響及其作用機(jī)制。 結(jié)果MTT結(jié)果表明DHA可降低3T3-L1前脂肪細(xì)胞的活力。對3T3-L1前脂肪細(xì)胞周期的研究發(fā)現(xiàn),DHA能誘導(dǎo)前脂肪細(xì)胞G2/M期阻滯,增強(qiáng)ERK1/2的活化、上調(diào)p21的表達(dá)。Hoechst染色顯示DHA可誘導(dǎo)3T3-L1前脂肪細(xì)胞凋亡,JC-1染色表明DHA可降低線粒體膜電位,蛋白免疫印跡分析發(fā)現(xiàn)DHA可上調(diào)凋亡相關(guān)蛋白p53表達(dá)、降低Bcl-2/Bax比值。同時(shí)發(fā)現(xiàn)DHA可增強(qiáng)前脂肪細(xì)胞凋亡效應(yīng)因子Caspase 3的活性。對成熟脂肪細(xì)胞的研究發(fā)現(xiàn),DHA可降低3T3-L1成熟脂肪細(xì)胞的活力、降低脂質(zhì)含量,Hoechst染色結(jié)果顯示DHA能誘導(dǎo)3T3-L1成熟脂肪細(xì)胞凋亡,JC-1染色表明DHA處理24h可降低成熟脂肪細(xì)胞的線粒體膜電位,蛋白免疫印跡分析發(fā)現(xiàn)DHA可降低Bcl-2/Bax比值、抑制成熟脂肪細(xì)胞抗凋亡蛋白Akt和ERK1/2的活化、抑制CREB表達(dá),上調(diào)促凋亡蛋白Bad水平,還發(fā)現(xiàn)DHA可增強(qiáng)成熟脂肪細(xì)胞凋亡效應(yīng)因子Caspase 3的活性。 結(jié)論DHA可激活ERK1/2/p21途徑誘導(dǎo)G2/M期阻滯,抑制3T3-L1前脂肪細(xì)胞增殖,并通過上調(diào)p53表達(dá)、降低Bcl-2/Bax比值、升高Caspase 3活性,誘導(dǎo)前脂肪細(xì)胞凋亡。DHA通過抑制Akt/CREB和ERK途徑、降低Bcl-2/Bax比值、上調(diào)促凋亡蛋白Bad水平、升高Caspase 3活性,誘導(dǎo)成熟脂肪細(xì)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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

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