本文選題：神經(jīng)母細(xì)胞瘤　切入點(diǎn)：自噬抑制劑3-MA　出處：《河北醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:研究通過抑制自噬,增強(qiáng)常規(guī)化療藥物順鉑對(duì)神經(jīng)母細(xì)胞瘤SH-SY5Y治療效果的可能性,并探究其可能的分子機(jī)制。方法:1將人神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞株進(jìn)行體外培養(yǎng),采用不同濃度(0、1.25、2.5、5、10、20、40ug/ml)的順鉑分別處理處于對(duì)數(shù)生長期的神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞24h、48h、72h,及不同濃度(0、1.25、2.5、5、10mM)的3-MA分別處理處于對(duì)數(shù)生長期的神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞24h后,應(yīng)用MTS比色法測定細(xì)胞的存活率,采用不同濃度(0、2.5、5、10、20ug/ml)的順鉑及聯(lián)合應(yīng)用自噬抑制劑3-MA(5mM)處理處于對(duì)數(shù)生長期的神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞24h后,應(yīng)用MTS比色法測定細(xì)胞的存活率,并分別計(jì)算單獨(dú)應(yīng)用順鉑及順鉑聯(lián)合自噬抑制劑后順鉑的IC50值。2于普通光學(xué)顯微鏡下觀察對(duì)照組、常規(guī)化療藥物順鉑(10ug/ml)組、自噬抑制劑3-MA(5mM)組、自噬抑制劑3-MA(5mM)聯(lián)合常規(guī)化療藥物順鉑(10ug/ml)組細(xì)胞形態(tài)學(xué)上的改變。3用同樣的方法處理細(xì)胞后,將四組處理后的細(xì)胞進(jìn)行PI染色,在熒光顯微鏡下觀察晚期凋亡及壞死細(xì)胞。4應(yīng)用western blot方法分別檢測上述四組細(xì)胞的自噬相關(guān)蛋白Beclin-1、LC3-Ⅱ/Ⅰ及凋亡相關(guān)蛋白Caspase-3的表達(dá)變化情況。實(shí)驗(yàn)數(shù)據(jù)應(yīng)用SPSS 13.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。計(jì)量資料均進(jìn)行正態(tài)性及方差齊性檢驗(yàn),經(jīng)檢驗(yàn)后符合正態(tài)分布的計(jì)量資料采用均數(shù)±標(biāo)準(zhǔn)差(x±S)表示,不符合正態(tài)分布的計(jì)量資料采用中位數(shù)(四分位數(shù)間距)(M(P25~P75))表示。經(jīng)檢驗(yàn)符合正態(tài)分布且方差齊的多組均數(shù)的比較應(yīng)用單因素方差分析(One-way ANOVA),多組間的兩兩比較采用SNK法(Student-Newman-Keuls);非正態(tài)分布的數(shù)據(jù)多組比較采用Kruskalwallis H檢驗(yàn),兩兩比較采用Mann-Whitney U檢驗(yàn)。以α=0.05為檢驗(yàn)標(biāo)準(zhǔn),以p0.05作為差別顯著性判斷標(biāo)準(zhǔn)。結(jié)果:1經(jīng)順鉑處理人神經(jīng)母細(xì)胞瘤sh-sy5y細(xì)胞24小時(shí)后,對(duì)細(xì)胞活力影響較小,僅在順鉑濃度到達(dá)10ug/ml時(shí),細(xì)胞存活率有所下降(p0.05)。順鉑作用48小時(shí)后,當(dāng)順鉑濃度到達(dá)5ug/ml時(shí),細(xì)胞活力下降(p0.01),當(dāng)順鉑濃度到達(dá)10ug/ml時(shí),細(xì)胞活力下降顯著(p0.001)。順鉑作用72小時(shí)后,除濃度為1.25ug/ml組,其余各組細(xì)胞活力均下降顯著,具有統(tǒng)計(jì)學(xué)意義(p0.05)。不同濃度的自噬抑制劑3-ma單獨(dú)作用于神經(jīng)母細(xì)胞瘤sh-sy5y細(xì)胞24h后,對(duì)細(xì)胞活力無明顯影響,即使3-ma在最大濃度10mmol/l時(shí),細(xì)胞存活率與空白對(duì)照組仍無統(tǒng)計(jì)學(xué)差異(p0.05)。單獨(dú)應(yīng)用順鉑及聯(lián)合自噬抑制劑3-ma后順鉑的ic50值分別為10.9ug/ml和5.25ug/ml,聯(lián)合自噬抑制劑后順鉑的ic50值降低了49%。2于光學(xué)顯微鏡下觀察細(xì)胞形態(tài)學(xué)的改變,發(fā)現(xiàn)對(duì)照組細(xì)胞數(shù)目較多,均貼壁生長,細(xì)胞形態(tài)較一致,呈長梭形或多角形,體積較小,有聚集成團(tuán)傾向,細(xì)胞代謝產(chǎn)物較少;自噬抑制劑處理組細(xì)胞數(shù)量無明顯減少,但細(xì)胞凸起不明顯,少量呈現(xiàn)橢圓形,順鉑處理組細(xì)胞數(shù)量明顯減少,形態(tài)發(fā)生較明顯變化,看不到長梭形狀的細(xì)胞,胞質(zhì)皺縮,多呈圓形或短橢圓形,聚集成團(tuán),自噬抑制劑聯(lián)合順鉑組細(xì)胞數(shù)量減少更明顯,且細(xì)胞分散,看不到明顯成團(tuán)細(xì)胞,培養(yǎng)皿內(nèi)可見大量細(xì)胞產(chǎn)物及死亡細(xì)胞。3熒光顯微鏡下觀察細(xì)胞的晚期凋亡及壞死情況發(fā)現(xiàn),對(duì)照組細(xì)胞生長良好,未見晚期凋亡及壞死細(xì)胞,3-ma處理組晚期凋亡及壞死細(xì)胞較對(duì)照組無明顯增多,順鉑處理組較對(duì)照組及3-ma處理組晚期凋亡及壞死細(xì)胞明顯增多,順鉑聯(lián)合3-ma處理組細(xì)胞的晚期凋亡及壞死率又明顯高于單用順鉑組。4westernblot檢測結(jié)果顯示:對(duì)照組自噬及凋亡相關(guān)蛋白均處于基礎(chǔ)水平,應(yīng)用3-ma抑制自噬后,檢測到自噬相關(guān)蛋白表達(dá)下降,但凋亡相關(guān)蛋白caspase-3表達(dá)并未見明顯增高;順鉑處理神經(jīng)母細(xì)胞瘤sh-sy5y細(xì)胞24h后,與對(duì)照組相比自噬相關(guān)蛋白beclin-1、lc3-Ⅱ/Ⅰ及凋亡相關(guān)蛋白caspase-3的表達(dá)均升高,加入自噬抑制劑后,自噬相關(guān)蛋白Beclin-1、LC3-Ⅱ/Ⅰ的表達(dá)較單獨(dú)應(yīng)用順鉑組明顯下降,但凋亡相關(guān)蛋白Caspase-3的表達(dá)較單獨(dú)應(yīng)用順鉑組明顯升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1順鉑能夠在體外抑制神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞的增值,且在一定的濃度范圍內(nèi)這種抑制作用具有時(shí)間、劑量依賴性;2單獨(dú)應(yīng)用自噬抑制劑3-MA 24小時(shí),在一定的濃度范圍內(nèi)無明顯抑制增值作用,即單獨(dú)抑制自噬并不能有效抑制神經(jīng)母細(xì)胞瘤的增殖;3順鉑聯(lián)合自噬抑制劑3-MA后,可降低順鉑的IC50值,起化療增敏作用;4順鉑誘導(dǎo)神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞發(fā)生凋亡的同時(shí),自噬作用增強(qiáng),且自噬在細(xì)胞凋亡過程中起保護(hù)作用;5抑制自噬可以加強(qiáng)順鉑誘導(dǎo)神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞凋亡的作用,增強(qiáng)其化療效果,提示自噬抑制劑可提高常規(guī)化療藥物順鉑對(duì)神經(jīng)母細(xì)胞瘤的治療效果,較單純常規(guī)化療更有價(jià)值,為神經(jīng)母細(xì)胞瘤的臨床治療提供了新的思路和方法。
[Abstract]:Objective: To study the inhibition of autophagy, enhance the possibility of conventional chemotherapy drug cisplatin on neuroblastoma SH-SY5Y treatment, and to explore its possible molecular mechanisms. Methods: 1 human neuroblastoma cell line SH-SY5Y were cultured in vitro, with different concentrations of cisplatin (0,1.25,2.5,5,10,20,40ug/ml) treatment in neuroblastoma SH-SY5Y cells 24h, logarithmic growth phase and different concentrations of 48h, 72h (0,1.25,2.5,5,10mM) were treated with 3-MA in neuroblastoma SH-SY5Y cells in logarithmic growth phase after 24h, colorimetric determination of cell survival rate by MTS, using different concentration of cisplatin (0,2.5,5,10,20ug/ml) and the combined application of autophagy inhibitor 3-MA (5mM) in the treatment of SH-SY5Y cells in logarithmic growth phase after 24h, colorimetric determination of cell survival rate by MTS, and calculated the separate application of cisplatin and cisplatin Combined with autophagy inhibitor after cisplatin IC50 value of.2 in ordinary optical microscope in control group, conventional chemotherapy drug cisplatin (10ug/ml) group, autophagy inhibitor 3-MA (5mM) group, autophagy inhibitor 3-MA (5mM) combined with conventional chemotherapy drug cisplatin (10ug/ml) group of cell morphological changes of.3 cells treated with the same method. The four groups of the treated cells were stained with PI, observed late apoptosis and necrosis were detected using western.4 blot of the four group of the autophagy related protein Beclin-1 in the fluorescence microscope, the expression of LC3- II / I and apoptosis related protein Caspase-3. The experimental data using SPSS 13 statistical software for statistical analysis. The measurement data were normality and homogeneity of variance test, after testing with normal distribution measurement data by the mean and standard deviation (x + S) said, does not conform to the normal distribution and measurement The median (four percentile interval) (M (P25~P75)). After testing in line with the analysis of the normal distribution and homogeneity of multiple mean comparison using single factor variance (One-way, ANOVA) - between 22 groups were compared with SNK method (Student-Newman-Keuls); non normal distribution data sets compared with Kruskalwallis H test, 22 compared with Mann-Whitney U test. A =0.05 test, with P0.05 as the difference significant criteria. Results: 1 after treatment with cisplatin in human neuroblastoma SH-SY5Y cells after 24 hours, the cell viability is less affected, only reached 10ug/ml in cisplatin concentration, cell survival rate decreased (P0.05). 48 hours after cisplatin, when cisplatin concentration reached 5ug/ml, decreased cell viability (P0.01), when the cisplatin concentration reached 10ug/ml, cell viability decreased significantly (p0.001). The effect of cisplatin for 72 hours, in addition to a concentration of 1.25 Ug/ml group, cell viability decreased significantly in other groups, with statistical significance (P0.05). Autophagy inhibitors with different concentrations of 3-mA alone in neuroblastoma SH-SY5Y cells after 24h had no obvious effect on cell viability, even if the 3-mA in the maximum concentration of 10mmol/l, the cell survival rate was no significant difference with the control group (P0.05). Cisplatin alone and in combination with autophagy inhibitor 3-mA after cisplatin IC50 values were 10.9ug/ml and 5.25ug/ml, IC50 reduced 49%.2 in cisplatin under optical microscope to observe the cell morphology changes value combined with autophagy inhibitor, was found in the control group had more number of cells were adherent growth, cell morphology was similar to that of spindle or polygonal, small volume, has aggregated tendency, cell metabolism is less; the number of cells without the autophagy inhibitor treatment group decreased significantly, but the cell protrusion is not obvious, a small present oval The shape, cisplatin treatment group significantly decreased, morphology significantly changed, can not see the long spindle shaped cells, cytoplasmic shrinkage, round or oval, together, the number of cell autophagy inhibitor combined with cisplatin group decreased significantly, and the cells dispersed, do not see a significant cluster of cells, culture there were a lot of dish products and death of.3 cells were observed under fluorescence microscope late apoptosis and necrosis of cells that controls cell growth is good, no late apoptotic and necrotic cells, 3-mA treatment group late apoptosis and necrosis than in control group significantly increased, cisplatin group compared with control group and 3-mA treatment group and late apoptosis necrotic cells increased significantly, cisplatin combined with 3-mA treatment group advanced cell apoptosis and necrosis rate was significantly higher than that of cisplatin group.4westernblot test results show that: the group of autophagy and apoptosis related protein At the basic level, the application of 3-mA after the inhibition of autophagy, detected the expression of autophagy related protein decreased, but the expression of apoptosis related protein caspase-3 was significantly increased; cisplatin treated neuroblastoma SH-SY5Y cells after 24h, compared with the control group the expression of autophagy related protein beclin-1, lc3- II / I and apoptosis related protein caspase-3 increased significantly. After joining the autophagy inhibitor, autophagy related protein Beclin-1, LC3- II / I. expression compared with cisplatin alone group decreased significantly, but the expression of apoptosis related protein Caspase-3 compared with cisplatin alone group was significantly increased, the difference was statistically significant (P0.05). Conclusion: 1 cisplatin can inhibit neuroblastoma SH-SY5Y cells in vitro added. And this in a certain concentration range inhibition is time dose dependent; 2 separate application of autophagy inhibitor 3-MA for 24 hours, in a certain concentration range without obvious The growth inhibition effect, which alone can not effectively inhibit autophagy and inhibit neuroblastoma proliferation; 3 cisplatin combined with autophagy inhibitor 3-MA, cisplatin can reduce the IC50 value on chemotherapy sensitization; neuroblastoma cell apoptosis induced by cisplatin 4 SH-SY5Y at the same time, enhance the apoptosis and autophagy plays a protective role. In the process of cell apoptosis; 5 inhibition of autophagy can enhance cisplatin induced apoptosis of neuroblastoma SH-SY5Y cells and enhance the effect of chemotherapy, suggesting that autophagy inhibitors may improve the therapeutic effect of conventional chemotherapy drug cisplatin on neuroblastoma cells, compared with conventional chemotherapy is more valuable, to provide new ideas and methods for clinical treatment of nerve blastomas.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.4

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