本文選題：卵巢透明細(xì)胞癌 + siRNA�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：研究目的：體外設(shè)計(jì)并合成靶向于人ARID1A基因的siRNA,轉(zhuǎn)染人卵巢透明細(xì)胞癌ES2細(xì)胞,篩選出下調(diào)ARID1A表達(dá)的有效片段,研究ARID1A基因沉默對(duì)順鉑敏感性的影響,探討可能涉及的分子通路改變；研究蛋白激酶B抑制劑哌立福辛聯(lián)合順鉑對(duì)ES2細(xì)胞的體外增殖抑制作用,探討二者聯(lián)合的協(xié)同作用及可能的促凋亡機(jī)制,為今后卵巢透明細(xì)胞癌的分子靶向治療提供一定的理論依據(jù)。研究方法：體外合成各組siRNA并瞬時(shí)轉(zhuǎn)染ES2細(xì)胞；利用RT-PCR法和western blot法分別從mRNA和蛋白水平檢測(cè)ARID1A基因沉默情況,篩選出最佳片段(即siRNA-3)。使用siRNA-3片段瞬時(shí)轉(zhuǎn)染ES2細(xì)胞,24 h后加入不同濃度的順鉑,孵育48 h后采用CCK-8法檢測(cè)各實(shí)驗(yàn)組半數(shù)抑制濃度(IC50)；轉(zhuǎn)染后24h加入相同濃度的順鉑50 μM,孵育48 h后測(cè)得各組細(xì)胞生存率；采用流式細(xì)胞儀檢測(cè)各實(shí)驗(yàn)組ES2細(xì)胞的凋亡率：采用western blot法檢測(cè)各實(shí)驗(yàn)組ES2細(xì)胞中蛋白激酶B(AKT)表達(dá)水平的變化。以不同濃度哌立福辛作用于ES2細(xì)胞48 h,采用CCK-8法檢測(cè)細(xì)胞增殖抑制情況；以哌立福辛最小有作用劑量與不同濃度順鉑聯(lián)合處理細(xì)胞48 h,以金氏公式篩選出協(xié)同抑制作用最明顯的聯(lián)合劑量,以該聯(lián)合劑量處理ES2細(xì)胞48 h,采用DAPI染色法及流式細(xì)胞儀檢測(cè)細(xì)胞的凋亡情況；采用Giemsa染色法檢測(cè)細(xì)胞有絲分裂指數(shù)；采用western blot法檢測(cè)細(xì)胞中Caspase-3(cleaved)及PARP(cleaved)蛋白表達(dá)水平的變化。研究結(jié)果：使用ARID1A特異性siRNA-3片段轉(zhuǎn)染ES2細(xì)胞后,CCK-8法結(jié)果顯示：siRNA-3組的IC50較siRNA-NC組和空白對(duì)照組均升高(均P0.05)；加入相同濃度的順鉑, siRNA-3組細(xì)胞生存率明顯高于siRNA-NC組和空白對(duì)照組(均P0.05)。流式結(jié)果顯示：siRNA-3聯(lián)合順鉑組細(xì)胞凋亡率明顯低于順鉑組(P0.01),siRNA-3組細(xì)胞凋亡率低于對(duì)照組(P0.05)；western blot結(jié)果顯示：siRNA-3組中AKT蛋白表達(dá)水平較對(duì)照組升高(P0.01),順鉑組中AKT蛋白表達(dá)水平較對(duì)照組降低(P0.01),siRNA-3聯(lián)合順鉑組中AKT蛋白表達(dá)水平較順鉑組升高(P0.05)。CCK-8法檢測(cè)顯示：哌立福辛在48 h對(duì)ES2細(xì)胞的最小有作用劑量為4μM,以金氏公式計(jì)算結(jié)果提示4 μM哌立福辛+40μM順鉑聯(lián)合用藥協(xié)同抑制細(xì)胞增殖作用最強(qiáng);與哌立福辛或順鉑單獨(dú)作用組相比,DAPI染色發(fā)現(xiàn)聯(lián)合用藥組鏡下可見(jiàn)大量的細(xì)胞核碎裂；聯(lián)合用藥能夠協(xié)同增加細(xì)胞凋亡率；協(xié)同抑制細(xì)胞有絲分裂；聯(lián)合用藥能夠明顯增加細(xì)胞中Caspase-3 (cleaved)蛋白的表達(dá),降低PARP(cleaved)蛋白的表達(dá)(P0.01)。研究結(jié)論：采用siRNA干擾片段沉默卵巢透明細(xì)胞癌ES2細(xì)胞中ARID1A基因表達(dá)后能夠降低ES2細(xì)胞對(duì)順鉑的敏感性,減少細(xì)胞的凋亡；推測(cè)其機(jī)制可能涉及蛋白激酶B(AKT)信號(hào)通路的激活。AKT抑制劑哌立福辛聯(lián)合順鉑能夠協(xié)同抑制卵巢透明細(xì)胞癌ES2細(xì)胞的增殖并誘導(dǎo)腫瘤細(xì)胞發(fā)生凋亡：其促凋亡機(jī)制可能是通過(guò)上調(diào)Caspase-3 (cleaved)及下調(diào)PARP(cleaved)蛋白的表達(dá)實(shí)現(xiàn)的。
[Abstract]:The aim of this study was to design and synthesize siRNA targeting human ARID1A gene in vitro , transfected human ovarian clear cell carcinoma ES2 cells , screened the effective fragment of down - regulated ARID1A expression , studied the effect of ARID1A gene silencing on cisplatin sensitivity , and discussed possible molecular pathways change ;
In vitro proliferation inhibition of ES2 cells was investigated by the combination of protein kinase B inhibitor and cisplatin in vitro . The synergistic effect and possible mechanism of apoptosis were discussed .
RT - PCR and western blot were used to detect the silencing of ARID1A gene from mRNA and protein levels respectively . The best fragment ( i.e . siRNA - 3 ) was screened . After 24 hours of transfection of ES2 cells with siRNA - 3 fragment , different concentrations of cisplatin were added . After 48 h incubation , median inhibitory concentration ( IC50 ) was determined by CCK - 8 method .
After transfection , the survival rate of each group was measured after 48 h incubation with the same concentration of cisplatin ( 50 渭M ) .
The apoptosis rate of ES2 cells in each experimental group was detected by flow cytometry . The expression level of protein kinase B was detected in ES2 cells of each experimental group by western blot .
Combined with cisplatin ( DDP ) treatment for 48 h with the minimum effective dose of piperacillin , the most obvious combination dose was screened by using the gold formula . The apoptosis of ES2 cells was treated with DAPI staining and flow cytometry .
The mitotic index of the cells was detected by Gigi staining .
The expression level of Caspase - 3 was detected by western blot . Results : After transfection of ES2 cells with ARID1A - specific siRNA - 3 fragment , CCK - 8 method showed that the IC50 of siRNA - 3 group was higher than that of siRNA - NC group and blank control group ( all P0.05 ) .
The survival rate of siRNA - 3 group was significantly higher than that in the control group ( P0.05 ) . The results showed that the apoptosis rate of siRNA - 3 combined with cisplatin group was significantly lower than that in the cisplatin group ( P0.01 ) , and the apoptosis rate of siRNA - 3 group was lower than that of control group ( P0.05 ) .
The results of western blot showed that the expression level in the siRNA - 3 group was higher than that in the control group ( P0.01 ) . The expression level in the cisplatin group was lower than that in the control group ( P0.01 ) .
the combination therapy can increase the cell apoptosis rate ;
Synergistic inhibition of cell mitosis ;
Combined use of siRNA could significantly increase the expression of Caspase - 3 protein in the cells and decrease the expression of parp ( P0.01 ) . Conclusion : The sensitivity of ES2 cells to cis - platinum can be reduced and the apoptosis of the cells can be reduced after the expression of ARID1A gene in ES2 cells of ovarian clear cell carcinoma is silenced by siRNA interference fragments .
It is speculated that the mechanism may involve the activation of the signaling pathway of protein kinase B ( PKC ) , which can synergistically inhibit the proliferation of ovarian clear cell carcinoma ES2 cells and induce apoptosis of tumor cells : its pro - apoptotic mechanism may be achieved by up - regulation of Caspase - 3 ( Caspase - 3 ) and down - regulation of the expression of parp - protein .

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R737.31

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本文編號(hào)：1846828