發(fā)布時(shí)間：2018-05-06 20:37

  本文選題：哇巴因 + Raji細(xì)胞��； 參考：《南京中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：[目的]研究強(qiáng)心苷類藥物哇巴因?qū)τ谌薆urkitt's淋巴瘤細(xì)胞株Raji細(xì)胞抑制增殖、誘導(dǎo)凋亡和自噬的作用及其作用機(jī)制。[方法]以不同濃度(25nM、50nM、100nM)哇巴因作用于Raji細(xì)胞,采用CCK-8法檢測(cè)哇巴因?qū)�Raji細(xì)胞和正常人骨髓單個(gè)核細(xì)胞(BM-MNCs)的增殖抑制作用;應(yīng)用光鏡和AnnexinV-FITC/PI雙染法觀察Raji細(xì)胞形態(tài),檢測(cè)細(xì)胞凋亡率,Western Blot法檢測(cè)cleaved-caspase-3、Bax、Bcl-2蛋白表達(dá)水平的變化;通過(guò)透射電鏡(TEM)、單丹磺酰戊二胺(MDC)染色法檢測(cè)Raji細(xì)胞自噬,Western Blot法檢測(cè)Beclin-1、LC3和PI3K/AKT/mTOR通路蛋白表達(dá)水平的變化。[結(jié)果]1.哇巴因呈濃度依賴性地抑制Raji細(xì)胞增殖。25 nM、50 nM、100nM哇巴因?qū)�Raji細(xì)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(xì)胞48h的IC50值為76.48nM。而同等濃度哇巴因?qū)θ薆M-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(xì)胞相比,抑制率均有顯著性差異(P0.05)。2.哇巴因可誘導(dǎo)Raji細(xì)胞凋亡。光鏡下可觀察到Raji細(xì)胞出現(xiàn)胞漿空泡化、細(xì)胞膜皺縮、染色質(zhì)增粗、濃集和邊聚等凋亡形態(tài)學(xué)改變;25 nM、50 nM、100 nM濃度的哇巴因作用Raji細(xì)胞48h后,Raji細(xì)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(xì)胞早期凋亡率與對(duì)照組無(wú)明顯差異外(P0.05),其余各實(shí)驗(yàn)組早期凋亡率、晚期凋亡率和總凋亡率與對(duì)照組相比,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。隨著哇巴因作用濃度的升高,cleaved-caspase-3蛋白的表達(dá)明顯增強(qiáng),Bax/Bcl-2比值增加,與對(duì)照組相比,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。3.哇巴因可誘導(dǎo)Raji細(xì)胞自噬。TTEM下可觀察到Raji細(xì)胞胞漿中出現(xiàn)許多雙層膜結(jié)構(gòu),包裹胞漿成分和線粒體等細(xì)胞器,形成自噬體、自噬溶酶體等典型的自噬形態(tài)學(xué)改變;MDC染色法可觀察到,以25nM、50nM、100nM哇巴因作用于Raji細(xì)胞,熒光染色的細(xì)胞數(shù)量隨作用濃度增加而逐漸增加,且細(xì)胞核周區(qū)域出現(xiàn)了較強(qiáng)的點(diǎn)塊狀熒光顆粒。隨著哇巴因作用濃度的升高,Beclin-1表達(dá)明顯增強(qiáng),LC3-II/LC3-I比值增加,與對(duì)照組相比,差異具有統(tǒng)計(jì)學(xué)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(shì),其中50nM和100nM哇巴因組與對(duì)照組相比,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。[結(jié)論]1.100 nM以下哇巴因?qū)�Raji細(xì)胞具有明顯增殖抑制作用,而對(duì)人BM-MNCs無(wú)明顯毒性。2.哇巴因?qū)�Raji細(xì)胞有誘導(dǎo)凋亡的作用,該過(guò)程伴隨著caspase-3活化和Bax/Bcl-2比值上調(diào)。3.哇巴因?qū)�Raji細(xì)胞有誘導(dǎo)自噬的作用,哇巴因可能通過(guò)負(fù)調(diào)控PBK/AKT/mTOR通路誘導(dǎo)Raji細(xì)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(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285

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