本文選題：哇巴因 + Raji細胞�。� 參考：《南京中醫(yī)藥大學》2017年博士論文

【摘要】：[目的]研究強心苷類藥物哇巴因對于人Burkitt's淋巴瘤細胞株Raji細胞抑制增殖、誘導凋亡和自噬的作用及其作用機制。[方法]以不同濃度(25nM、50nM、100nM)哇巴因作用于Raji細胞,采用CCK-8法檢測哇巴因對Raji細胞和正常人骨髓單個核細胞(BM-MNCs)的增殖抑制作用;應用光鏡和AnnexinV-FITC/PI雙染法觀察Raji細胞形態(tài),檢測細胞凋亡率,Western Blot法檢測cleaved-caspase-3、Bax、Bcl-2蛋白表達水平的變化;通過透射電鏡(TEM)、單丹磺酰戊二胺(MDC)染色法檢測Raji細胞自噬,Western Blot法檢測Beclin-1、LC3和PI3K/AKT/mTOR通路蛋白表達水平的變化。[結果]1.哇巴因呈濃度依賴性地抑制Raji細胞增殖。25 nM、50 nM、100nM哇巴因對Raji細胞的增殖抑制率分別為23.23±2.20%、32.20±4.09%和58.86±5.78%,各濃度組的抑制率具有顯著性差異(P0.05)。哇巴因作用于Raji細胞48h的IC50值為76.48nM。而同等濃度哇巴因對人BM-NNCs的增殖抑制率分別為5.44±0.91%、7.86±1.26%和17.31±1.55%,與Raji細胞相比,抑制率均有顯著性差異(P0.05)。2.哇巴因可誘導Raji細胞凋亡。光鏡下可觀察到Raji細胞出現(xiàn)胞漿空泡化、細胞膜皺縮、染色質增粗、濃集和邊聚等凋亡形態(tài)學改變;25 nM、50 nM、100 nM濃度的哇巴因作用Raji細胞48h后,Raji細胞早期凋亡率、晚期凋亡率和總凋亡率總體隨著哇巴因作用濃度的升高而增高,僅100nM哇巴因組早期凋亡率低于50 nM哇巴因組;除25 nM哇巴因組Raji細胞早期凋亡率與對照組無明顯差異外(P0.05),其余各實驗組早期凋亡率、晚期凋亡率和總凋亡率與對照組相比,差異均有統(tǒng)計學意義(P0.05)。隨著哇巴因作用濃度的升高,cleaved-caspase-3蛋白的表達明顯增強,Bax/Bcl-2比值增加,與對照組相比,差異具有統(tǒng)計學意義(P0.05)。3.哇巴因可誘導Raji細胞自噬。TTEM下可觀察到Raji細胞胞漿中出現(xiàn)許多雙層膜結構,包裹胞漿成分和線粒體等細胞器,形成自噬體、自噬溶酶體等典型的自噬形態(tài)學改變;MDC染色法可觀察到,以25nM、50nM、100nM哇巴因作用于Raji細胞,熒光染色的細胞數量隨作用濃度增加而逐漸增加,且細胞核周區(qū)域出現(xiàn)了較強的點塊狀熒光顆粒。隨著哇巴因作用濃度的升高,Beclin-1表達明顯增強,LC3-II/LC3-I比值增加,與對照組相比,差異具有統(tǒng)計學意義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趨勢,其中50nM和100nM哇巴因組與對照組相比,差異具有統(tǒng)計學意義(P0.05)。[結論]1.100 nM以下哇巴因對Raji細胞具有明顯增殖抑制作用,而對人BM-MNCs無明顯毒性。2.哇巴因對Raji細胞有誘導凋亡的作用,該過程伴隨著caspase-3活化和Bax/Bcl-2比值上調。3.哇巴因對Raji細胞有誘導自噬的作用,哇巴因可能通過負調控PBK/AKT/mTOR通路誘導Raji細胞發(fā)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 鹵2.20 鹵4.09% and 58.86 鹵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 鹵1.26% and 17.31 鹵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.
【學位授予單位】：南京中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R285

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