Daxx介导ox-LDL诱导巨噬细胞凋亡与胆固醇蓄积的关系

发布时间：2018-01-06 07:32

本文关键词：Daxx介导ox-LDL诱导巨噬细胞凋亡与胆固醇蓄积的关系　出处：《南华大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的：研究Daxx介导ox-LDL诱导巨噬细胞凋亡与胆固醇蓄积的关系。方法：用不同浓度的氧化型低密度脂蛋白(ox-LDL)处理RAW264.7细胞48h，MTT法检测ox-LDL对RAW264.7细胞生长的影响；流式细胞术和DNA断裂片段分析法研究ox-LDL诱导的细胞凋亡；用特异性siRNA沉默Daxx在RAW264.7细胞中的表达，通过AO／EB染色观察基因沉默后的细胞凋亡形态学改变；高效液相色谱(HPLC)检测细胞内胆固醇含量；油红O染色观察细胞内脂滴的形成情况；Real time RT-PCR检测细胞内Daxx mRNA、SCAP mRNA的表达情况；用Western Blot印迹法检测caveolin-1蛋白的表达。结果：经不同浓度ox-LDL(0 mg／L、12.5 mg／L、25 mg／L、50 mg／L、75 mg／L)处理RAW264.7细胞48h后，MTT结果显示：随着药物浓度的增加，细胞活性明显下降，依次为100％±7.28％；103.2％±16.35％；76.27％±16.54％；56.49％±11.94％；40.65％±8.76％，ox-LDL在25mg／L时就对细胞活性产生明显的抑制作用，IC_(50)=50.6mg／L。流式细胞术结果显示：对照组细胞凋亡率为2.3％，用12.5 mg／L、25 mg／L、50 mg／L、75 mg／L ox-LDL处理RAW264.7细胞48h，凋亡率分别为4.9％、8.6％、14.7％、33.1％，凋亡率呈明显的浓度依赖性变化。此外，50 mg／L ox-LDL处理细胞不同时间(0h、12h、24h、48h、72h)，随着ox-LDL作用时间的延长，细胞凋亡率呈时间依赖性增加，50mg／L ox-LDL处理72h组凋亡率为27.9％。同时通过琼脂糖凝胶电泳分析DNA断片，50mg／L ox-LDL处理细胞48h组可观察到凋亡特征性DNA梯形条带。Real time RT-PCR结果发现随着ox-LDL浓度的递增和作用时间的延长，Daxx mRNA的相对表达量呈浓度依赖性和时间依赖性增加。在50 mg／L ox-LDL处理细胞48h时Daxx mRNA的相对表达量达到最高(Daxx／β-actin mRNA由1.25×10~(-4)上升至5.3×10~(-4))。RAW264.7细胞经50mg／L ox-LDL处理48h后，AO／EB染色显示明显的细胞凋亡形态学特征，出现核固缩、碎裂，同时细胞内胆固醇含量显著增加(由76.3μg／mg蛋白上升至368.5μg／mg蛋白)，油红O显示细胞内有大量的脂滴存在。用特异性siRNA沉默Daxx在RAW264.7细胞中的表达能引起凋亡抑制，AO／EB染色显示细胞凋亡率明显降低，同时细胞内胆固醇含量明显下降(由368.5μg／mg蛋白下降至236.9μg／mg蛋白)，油红O染色显示细胞内脂滴也明显减少；Western-blot显示caveolin-1表达呈时间依赖性上调，转染Daxx siRNA后其表达有所下调；Real time RT-PCR结果表明ox-LDL抑制SCAP mRNA的表达，经RNA干扰后，SCAP mRNA的表达上调。结论：1、Daxx具有介导ox-LDL诱导的RAW264.7巨噬细胞凋亡作用。 2、Daxx可能通过下调SCAP的表达和上调caveolin-1的表达来影响巨噬细胞源性荷脂细胞胆固醇的摄取，从而发挥促细胞凋亡作用。
[Abstract]:Aim: to study the relationship between Daxx-mediated ox-LDL-induced macrophage apoptosis and cholesterol accumulation. Methods: RAW264.7 cells were treated with oxidized low density lipoprotein (ox-LDL) for 48 h. The effect of ox-LDL on the growth of RAW264.7 cells was detected by MTT assay. Flow cytometry and DNA fragment analysis were used to study the apoptosis induced by ox-LDL. The expression of Daxx in RAW264.7 cells was silenced by specific siRNA. The morphological changes of apoptosis after gene silencing were observed by AO/EB staining. High performance liquid chromatography (HPLC) was used to detect the content of cholesterol in the cells. The formation of lipid droplets was observed by oil red O staining. The expression of Daxx mRNA-SCAP mRNA was detected by Real time RT-PCR. The expression of caveolin-1 protein was detected by Western Blot blot. Results: after different concentrations of ox-LDL(0 mg / L ~ (12.5) mg / L ~ (25) mg / L ~ (50) mg/L. RAW264.7 cells were treated with 75 mg / L for 48 h. The results showed that with the increase of drug concentration, the cell activity decreased significantly (100% 卤7.28). 103.2% 卤16.35; 76.27% 卤16.54; 56.49% 卤11.94; At 25 mg / L, 40.65% 卤8.76% ox-LDL significantly inhibited cell activity. The results of flow cytometry showed that the apoptotic rate of the control group was 2.3, with 12.5 mg / L ~ (25 mg / L) of 25 mg / L ~ (50) mg/L. When 75 mg/L ox-LDL was treated with RAW264.7 cells for 48 h, the apoptotic rate was 4.9% and 8.6%, respectively. The apoptosis rate was in a dose-dependent manner. In addition, the cells were treated with 50 mg/L ox-LDL for 12 h, 24 h, 48 h and 72 h). With the prolongation of ox-LDL, the apoptosis rate increased in a time-dependent manner. The apoptotic rate of 50 mg / L ox-LDL treated for 72 h was 27.9.The DNA fragment was analyzed by agarose gel electrophoresis. Apoptosis characteristic DNA trapezoid band. Real time was observed in cells treated with 50 mg / L ox-LDL for 48 h. The results of RT-PCR showed that with the increase of ox-LDL concentration and the prolongation of action time. The relative expression of Daxx mRNA was increased in a concentration-and time-dependent manner. Daxx was treated with 50 mg/L ox-LDL for 48 h. The relative expression of mRNA is the highest (. Daxx / 尾 -actin mRNA increased from 1.25 脳 10 ~ (-4) to 5.3 脳 10 ~ (-4)). RAW264.7 cells were treated with 50 mg / L ox-LDL for 48 h. AO/EB staining showed obvious morphological characteristics of apoptosis, nuclear pyknosis and fragmentation. At the same time, the content of cholesterol increased significantly (from 76.3 渭 g / mg protein to 368.5 渭 g / mg protein). Oil red O showed a large number of lipid droplets in the cells. Silencing the expression of Daxx in RAW264.7 cells with specific siRNA could induce apoptosis inhibition. AO/EB staining showed that the apoptosis rate and cholesterol content were significantly decreased (from 368.5 渭 g / mg protein to 236.9 渭 g / mg protein). Oil red O staining showed that lipid droplets also decreased significantly. Western-blot showed that the expression of caveolin-1 was up-regulated in a time-dependent manner, and its expression was down-regulated after transfection of Daxx siRNA. The results of Real time RT-PCR showed that ox-LDL inhibited the expression of SCAP mRNA and up-regulated the expression of SCAP mRNA after RNA interference. Conclusion Daxx can mediate the apoptosis of RAW264.7 macrophages induced by ox-LDL. 2Daxx may affect cholesterol uptake of macrophage derived lipids by down-regulating the expression of SCAP and up-regulating the expression of caveolin-1, thus promoting apoptosis.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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