發(fā)布時(shí)間：2018-03-14 18:08

  本文選題：鹽霉素　切入點(diǎn)：腎上腺皮質(zhì)癌　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:探討鹽霉素在體內(nèi)外對(duì)腎上腺皮質(zhì)癌SW13細(xì)胞的增殖、凋亡、周期、侵襲的影響及其作用機(jī)制。方法:以濃度為30、20、10、5、2.5、1.25、0.625、0μmol/L的鹽霉素處理SW13細(xì)胞,分別在24、48、72、96h時(shí)用Cell Counting Kit-8(CCK-8)試劑盒檢測(cè)其對(duì)增殖的影響并計(jì)算半抑制濃度(IC50)。流式細(xì)胞儀檢測(cè)鹽霉素處理后SW13細(xì)胞的周期及凋亡。通過(guò)AO-EB染色在熒光顯微鏡下觀察鹽霉素處理后SW13細(xì)胞的凋亡情況。劃痕實(shí)驗(yàn)檢測(cè)鹽霉素對(duì)SW13細(xì)胞遷移能力的影響。Transwell侵襲實(shí)驗(yàn)檢測(cè)鹽霉素對(duì)SW13細(xì)胞侵襲力的影響。蛋白免疫印記法檢測(cè)β-鏈蛋白(β-catenin)與B細(xì)胞淋巴瘤/白血病-2蛋白(Bcl-2)的表達(dá)。體內(nèi)實(shí)驗(yàn)觀察鹽霉素對(duì)裸鼠移植瘤生長(zhǎng)的抑制情況。TUNNEL實(shí)驗(yàn)檢測(cè)鹽霉素在體內(nèi)對(duì)SW13細(xì)胞凋亡的影響。免疫組化實(shí)驗(yàn)觀察鹽霉素在體內(nèi)對(duì)SW13移植瘤PCNA、Bcl-2、β-catenin蛋白表達(dá)水平的影響。結(jié)果:CCK-8結(jié)果顯示0～30μmol/L的鹽霉素對(duì)SW13細(xì)胞的抑制呈明顯的時(shí)間-計(jì)量依賴(lài)效應(yīng),24、48、72、96h對(duì)應(yīng)的IC50依次為25.65、7.97、2.89、0.40μmol/L。流式檢測(cè)結(jié)果顯示,鹽霉素處理SW13細(xì)胞后處于G1期的細(xì)胞百分比分別為(66.250±1.162)%、(71.693±0.849)%,與對(duì)照組(59.837±3.043)%相比差異具有統(tǒng)計(jì)學(xué)意義(P0.05);凋亡率分別為9.9%、30.3%、3.4%,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。AO-EB染色在熒光顯微鏡下觀察鹽霉素處理后SW13細(xì)胞的凋亡率較對(duì)照組明顯升高。細(xì)胞劃痕實(shí)驗(yàn)結(jié)果顯示鹽霉素SW13細(xì)胞的遷移能力下降。Transwell結(jié)果顯示對(duì)照組與實(shí)驗(yàn)組細(xì)胞侵襲數(shù)分別為66.800±5.263、30.400±2.881、17.600±3.050,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。蛋白質(zhì)免疫印跡結(jié)果顯示W(wǎng)nt/β-catenin信號(hào)通路相關(guān)蛋白β-catenin及抑制凋亡相關(guān)蛋白Bcl-2表達(dá)下調(diào)。體內(nèi)實(shí)驗(yàn)結(jié)果表明鹽霉素可抑制裸鼠移植瘤的生長(zhǎng)。TUNNEL實(shí)驗(yàn)表明鹽霉素在體內(nèi)也可促進(jìn)SW13細(xì)胞的凋亡。移植瘤免疫組化實(shí)驗(yàn)結(jié)果表明鹽霉素在體內(nèi)可降低SW13移植瘤PCNA、Bcl-2、β-catenin蛋白表達(dá)水平。結(jié)論:鹽霉素在體內(nèi)外可抑制腎上腺皮質(zhì)癌SW13細(xì)胞的增殖,阻滯其周期,減弱侵襲力并誘導(dǎo)凋亡,其機(jī)制與干擾Wnt/β-catenin信號(hào)通路及下調(diào)PCNA及凋亡抑制蛋白Bcl-2有關(guān)。
[Abstract]:Objective: to investigate the effects of salinomycin on the proliferation, apoptosis, cycle and invasion of adrenal cortical carcinoma (SW13) cells in vitro and in vivo. The effect of Cell Counting Kit-8 CCK-8 on proliferation was detected by Cell Counting Kit-8 CCK-8 kit and the semi-inhibitory concentration was calculated at 96 h. Flow cytometry was used to detect the cell cycle and apoptosis of SW13 cells treated with salinomycin. The AO-EB staining was used to observe the effect of salinomycin on the proliferation of SW13 cells. The effect of salinomycin on the migration of SW13 cells was detected by scratch assay. The effect of salinomycin on the invasiveness of SW13 cells was detected by transwell invasion assay. The 尾 -catenin and B thin cells were detected by protein imprinting assay. The effect of salinomycin on the growth of xenografts in nude mice was observed in vivo. The effect of salinomycin on the apoptosis of SW13 cells in vivo was detected by Tunel assay. The effect of salinomycin on the apoptosis of SW13 cells was observed by immunohistochemistry. Results the inhibitory effect of 0 ~ 30 渭 mol/L salinomycin on SW13 cells was time-metrological dependent in vivo, and the corresponding IC50 for 96 h was 25.657.97.72.89 渭 mol / L and 0.40 渭 mol 路L ~ (-1), respectively. The percentage of cells in G1 phase of SW13 treated with salinomycin was 66.250 鹵1.162 and 71.693 鹵0.849, respectively, which was significantly higher than that of control group (59.837 鹵3.043%), and the apoptotic rate was 9.90.33.4.The difference was statistically significant under fluorescence microscope. The cell scratch test showed that the migration ability of salinomycin SW13 cells decreased. Transwell results showed that the number of cell invasion in the control group and the experimental group was 66.800 鹵5.263 and 30.400 鹵2.881or 17.600 鹵3.050, respectively. The difference was statistically significant. Wnt/ 尾 -catenin signaling pathway related protein 尾 -catenin and inhibiting apoptosis-related protein Bcl-2 expression were down-regulated. In vivo, salinomycin could inhibit the growth of xenografts in nude mice. The results of immunohistochemistry showed that salinomycin could decrease the expression of PCNA Bcl-2, 尾 -catenin protein in SW13 xenografts. Conclusion: salinomycin can inhibit the proliferation of SW13 cells in adrenal cortical carcinoma in vivo and in vitro. The mechanism is related to the interference of Wnt/ 尾 -catenin signaling pathway and down-regulation of PCNA and Bcl-2.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R736.6

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