哇巴因诱导Raji细胞凋亡和自噬机制的研究

发布时间：2018-05-06 20:37

本文选题：哇巴因 + Raji细胞　；参考：《南京中医药大学》2017年博士论文

【摘要】：[目的]研究强心苷类药物哇巴因对于人Burkitt's淋巴瘤细胞株Raji细胞抑制增殖、诱导凋亡和自噬的作用及其作用机制。[方法]以不同浓度(25nM、50nM、100nM)哇巴因作用于Raji细胞,采用CCK-8法检测哇巴因对Raji细胞和正常人骨髓单个核细胞(BM-MNCs)的增殖抑制作用;应用光镜和AnnexinV-FITC/PI双染法观察Raji细胞形态,检测细胞凋亡率,Western Blot法检测cleaved-caspase-3、Bax、Bcl-2蛋白表达水平的变化;通过透射电镜(TEM)、单丹磺酰戊二胺(MDC)染色法检测Raji细胞自噬,Western Blot法检测Beclin-1、LC3和PI3K/AKT/mTOR通路蛋白表达水平的变化。[结果]1.哇巴因呈浓度依赖性地抑制Raji细胞增殖。25 nM、50 nM、100nM哇巴因对Raji细胞的增殖抑制率分别为23.23±2.20%、32.20±4.09%和58.86±5.78%,各浓度组的抑制率具有显著性差异(P0.05)。哇巴因作用于Raji细胞48h的IC50值为76.48nM。而同等浓度哇巴因对人BM-NNCs的增殖抑制率分别为5.44±0.91%、7.86±1.26%和17.31±1.55%,与Raji细胞相比,抑制率均有显著性差异(P0.05)。2.哇巴因可诱导Raji细胞凋亡。光镜下可观察到Raji细胞出现胞浆空泡化、细胞膜皱缩、染色质增粗、浓集和边聚等凋亡形态学改变;25 nM、50 nM、100 nM浓度的哇巴因作用Raji细胞48h后,Raji细胞早期凋亡率、晚期凋亡率和总凋亡率总体随着哇巴因作用浓度的升高而增高,仅100nM哇巴因组早期凋亡率低于50 nM哇巴因组;除25 nM哇巴因组Raji细胞早期凋亡率与对照组无明显差异外(P0.05),其余各实验组早期凋亡率、晚期凋亡率和总凋亡率与对照组相比,差异均有统计学意义(P0.05)。随着哇巴因作用浓度的升高,cleaved-caspase-3蛋白的表达明显增强,Bax/Bcl-2比值增加,与对照组相比,差异具有统计学意义(P0.05)。3.哇巴因可诱导Raji细胞自噬。TTEM下可观察到Raji细胞胞浆中出现许多双层膜结构,包裹胞浆成分和线粒体等细胞器,形成自噬体、自噬溶酶体等典型的自噬形态学改变;MDC染色法可观察到,以25nM、50nM、100nM哇巴因作用于Raji细胞,荧光染色的细胞数量随作用浓度增加而逐渐增加,且细胞核周区域出现了较强的点块状荧光颗粒。随着哇巴因作用浓度的升高,Beclin-1表达明显增强,LC3-II/LC3-I比值增加,与对照组相比,差异具有统计学意义CP0.05);PI3K/AKT/mTOR通路蛋白及其下游底物S6K1和4EBP1蛋白磷酸化水平呈下降趋势,其中50nM和100nM哇巴因组与对照组相比,差异具有统计学意义(P0.05)。[结论]1.100 nM以下哇巴因对Raji细胞具有明显增殖抑制作用,而对人BM-MNCs无明显毒性。2.哇巴因对Raji细胞有诱导凋亡的作用,该过程伴随着caspase-3活化和Bax/Bcl-2比值上调。3.哇巴因对Raji细胞有诱导自噬的作用,哇巴因可能通过负调控PBK/AKT/mTOR通路诱导Raji细胞发生自噬性死亡。
[Abstract]:[objective] to study the effect of ouabain on inhibiting proliferation, inducing apoptosis and autophagy of human Burkitt's lymphoma cell line Raji and its mechanism. [methods] the proliferation inhibition of ouabain on Raji cells and normal human bone marrow mononuclear cells (BM-MNCs) was detected by CCK-8 method, and the morphology of Raji cells was observed by light microscopy and AnnexinV-FITC/PI double staining. The expression of Bcl-2 protein was detected by Western Blot, and the protein expression of Beclin-1mc3 and PI3K/AKT/mTOR pathway in Raji cells was detected by transmission electron microscopy (TEM) and monosulfonyl succinylenediamine (MDC) staining. [result] 1. Ouabain inhibited the proliferation of Raji cells in a concentration-dependent manner. The inhibitory rates of ouabain on the proliferation of Raji cells were 23.23 卤2.20 卤4.09% and 58.86 卤5.78%, respectively. The IC50 value of ouabain treated Raji cells for 48 h was 76.48 nm. The inhibitory rates of ouabain at the same concentration on human BM-NNCs proliferation were 7.86 卤1.26% and 17.31 卤1.55%, respectively, which were significantly different from those of Raji cells. Ouabain can induce apoptosis of Raji cells. Under the light microscope, cytoplasmic vacuolation, cell membrane shrinkage, chromatin thickening, thickening and edge aggregation of Raji cells were observed. The apoptotic morphological changes such as concentration and edge aggregation of ouabain at the concentration of 25nM ~ 50nM ~ (100) nm could be observed in the early stage of apoptosis of Raji cells treated with ouabain for 48 h. The late apoptosis rate and total apoptosis rate increased with the increase of ouabain concentration, but the early apoptosis rate of 100nM ouabain group was lower than that of 50nM ouabain group. With the exception of 25nm ouabain group, the early apoptosis rate of Raji cells was not significantly different from that of the control group, but the other experimental groups had significant difference in early apoptosis rate, late apoptosis rate and total apoptosis rate compared with the control group. With the increase of ouabain concentration, the expression of cleaved-caspase-3 protein increased significantly, and the ratio of Bax-Bcl-2 increased. The difference was statistically significant compared with the control group. Ouabain induced autophagy of Raji cells. It was observed that there were many double-layer membrane structures in the cytoplasm of Raji cells, which encapsulated the cytoplasmic components and mitochondria to form autophagy. Typical morphologic changes of autophagy, such as autophagy lysosome, can be observed by MDC staining. The number of fluorescent staining cells increased with the increase of the concentration of Raji cells treated with 25nM ~ 50nM / 100nM ouabain. And there were strong spot block fluorescent granules in the perinuclear region. With the increase of ouabain concentration, the expression of Beclin-1 increased significantly, and the ratio of LC3-II / LC3-I increased significantly. Compared with the control group, the phosphorylation level of PI3K / AKTOR pathway protein and its downstream substrate S6K1 and 4EBP1 protein decreased significantly compared with the control group. The difference between 50nM and 100nM ouabain group was statistically significant compared with the control group (P 0.05). [conclusion] ouabain below 1.100 nm has obvious inhibitory effect on Raji cell proliferation, but has no obvious toxicity on human BM-MNCs. Ouabain can induce apoptosis in Raji cells, which is accompanied by the activation of caspase-3 and the upregulation of Bax/Bcl-2 ratio. 3. Ouabain can induce autophagy in Raji cells. Ouabain may induce autophagic death of Raji cells through negative regulation of PBK/AKT/mTOR pathway.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285

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