本文選題：PIK3CA基因　切入點：基因沉默　出處：《南昌大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:1.通過檢測阿司匹林(ASA)對人骨髓瘤細胞株RPMI-8226細胞的PIK3CA基因及蛋白表達的影響,探討PIK3CA基因作為ASA抗骨髓瘤作用靶點的可行性。2.通過沉默PIK3CA基因后檢測ASA對RPMI-8226細胞增殖活性的影響,及其下游效應(yīng)蛋白pAKT的變化,探討PIK3CA基因在ASA抗骨髓瘤效應(yīng)中的作用及分子機制。方法:1.采用CCK-8方法,檢測不同濃度ASA(0、2.5、5、10mmol/L)處理RPMI-8226細胞24h、48h、72h后細胞增殖變化。2.采用實時熒光定量PCR和Western blot法檢測不同濃度ASA對RPMI-8226細胞PIK3CA基因及蛋白表達水平。3.利用siRNA干擾技術(shù),將PIK3CAsiRNA轉(zhuǎn)染RPMI-8226細胞,熒光顯微鏡下觀測細胞轉(zhuǎn)染效果,實時熒光定量PCR檢測PIK3CA m RNA表達以檢測基因沉默效果。4.采用CCK-8方法,檢測沉默PIK3CA基因后,ASA處理后RPMI-8226細胞增殖抑制變化。5.采用流式細胞儀,檢測沉默PIK3CA基因后,ASA處理后RPMI-8226細胞凋亡與周期變化。6.采用Western blot法,檢測沉默PIK3CA基因后,ASA處理后RPMI-8226細胞AKT與pAKT蛋白表達的變化。結(jié)果:1.在2.5~10mmol/L范圍內(nèi),ASA以劑量依賴性抑制骨髓瘤細胞株RPMI-8226細胞增殖。在24~72h內(nèi),ASA對RPMI-8226細胞的增殖抑制率呈時間依賴性。2.在2.5~10mmol/L范圍內(nèi),ASA以劑量依賴性抑制RPMI-8226細胞的PIK3CA基因與蛋白表達。3.PIK3CAsiRNA轉(zhuǎn)染RPMI-8226細胞后,PIK3CA基因表達下降83.2%,表明基因沉默效果較好。4.與對照組相比,PIK3CAsiRNA轉(zhuǎn)染后,ASA對RPMI-8226細胞的抗增殖活性明顯下降,且并不隨ASA濃度增大而變化。5.與對照組相比,PIK3CAsiRNA轉(zhuǎn)染后,ASA對RPMI-8226細胞的凋亡誘導(dǎo)活性明顯下降。6.與對照組相比,PIK3CAsiRNA轉(zhuǎn)染后,ASA對RPMI-8226細胞的G1期阻滯作用明顯下降。7.ASA以劑量依賴性抑制RPMI-8226細胞pAKT蛋白表達,但不影響總AKT蛋白表達。沉默PIK3CA基因后,ASA對RPMI-8226細胞pAKT蛋白抑制作用明顯減弱。結(jié)論:1.ASA以濃度依賴性抑制骨髓瘤細胞株RPMI-8226細胞的PIK3CA基因及蛋白表達。2.PIK3CA基因沉默可明顯減弱ASA對RPMI-8226細胞的增殖抑制、凋亡誘導(dǎo)效應(yīng)及細胞周期G1期阻滯作用。3.沉默PIK3CA基因顯著降低ASA對RPMI-8226細胞AKT磷酸化的抑制作用。4.ASA通過抑制PIK3CA基因及下游效應(yīng)分子AKT磷酸化而起抗骨髓瘤作用。
[Abstract]:Objective to investigate the effect of aspirin on the expression of PIK3CA gene and protein in human myeloma cell line RPMI-8226. To explore the feasibility of PIK3CA gene as a target of ASA anti-myeloma. (2) to detect the effect of ASA on the proliferation of RPMI-8226 cells and the change of downstream effector protein pAKT by silencing PIK3CA gene. To investigate the role and molecular mechanism of PIK3CA gene in the anti-myeloma effect of ASA. The proliferation of RPMI-8226 cells was detected after treatment with different concentrations of ASA for 24 h or 48 h or 72 h. Real-time quantitative PCR and Western blot were used to detect the PIK3CA gene and protein expression level of RPMI-8226 cells with different concentrations of ASA. PIK3CAsiRNA was transfected into RPMI-8226 cells by siRNA interference technique. The effect of cell transfection was observed under fluorescence microscope, and the expression of PIK3CA m RNA was detected by real-time fluorescence quantitative PCR to detect the effect of gene silencing. 4. CCK-8 method was used to detect the inhibition of RPMI-8226 cell proliferation after PIK3CA gene silencing. 5. Flow cytometry was used. Apoptosis and cell cycle changes of RPMI-8226 cells treated with silencing PIK3CA gene were detected. 6. Western blot assay was used to detect apoptosis and cell cycle change of RPMI-8226 cells. The changes of AKT and pAKT protein expression in RPMI-8226 cells treated with silencing PIK3CA gene were detected. Results 1. The proliferation of RPMI-8226 cells was inhibited in a dose-dependent manner in the range of 2.510 mmol / L. The inhibitory rate of AKT and pAKT protein on RPMI-8226 cells was observed within 2472 h. In a dose-dependent manner, the expression of PIK3CA gene and protein in RPMI-8226 cells was inhibited in a dose-dependent manner. 3. PIK3CAsiRNA transfection into RPMI-8226 cells decreased the expression of PIK3CA gene by 83.2%, which indicated that gene silencing effect was better than that of control group. The antiproliferative activity of RPMI-8226 cells decreased significantly. Compared with the control group, the apoptosis-inducing activity of PIK3CAsiRNA on RPMI-8226 cells decreased significantly. 6. Compared with the control group, the G 1 phase arrest of RPMI-8226 cells was significantly decreased by PIK3CAsiRNA transfection. The expression of pAKT protein in RPMI-8226 cells was inhibited in a dependent manner. After silencing the PIK3CA gene, the inhibition of pAKT protein in RPMI-8226 cells was obviously weakened. Conclusion: 1. The inhibition of PIK3CA gene and protein expression in RPMI-8226 cell line by ASA in a concentration dependent manner. 2. PIK3CA gene silencing can make it clear. The inhibitory effect of ASA on the proliferation of RPMI-8226 cells was significantly decreased. Apoptosis-induced effect and G1 phase arrest of cell cycle .3. silencing PIK3CA gene significantly reduced the inhibitory effect of ASA on AKT phosphorylation in RPMI-8226 cells. 4. ASA played an anti-myeloma effect by inhibiting the phosphorylation of PIK3CA gene and downstream effector molecule AKT.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R733.3

【參考文獻】

相關(guān)期刊論文 前3條

1 周新科;何璐;梁敏;劉季芳;;殼聚糖納米粒介導(dǎo)的PIK3CA基因干擾對胃癌細胞侵襲轉(zhuǎn)移能力的影響[J];南方醫(yī)科大學(xué)學(xué)報;2014年10期

2 Roberto Ria;Antonia Reale;Angelo Vacca;;Novel agents and new therapeutic approaches for treatment of multiple myeloma[J];World Journal of Methodology;2014年02期

3 劉恩菊 ,項永兵 ,金凡 ,周淑貞 ,孫璐 ,方茹蓉 ,阮志賢 ,高立峰 ,高玉堂;上海市區(qū)惡性腫瘤發(fā)病趨勢分析(1972～1999年)[J];腫瘤;2004年01期

相關(guān)博士學(xué)位論文 前1條

1 丁江華;阿司匹林對多發(fā)性骨髓瘤的抗腫瘤效應(yīng)及其增強硼替佐米細胞毒作用的分子機制研究[D];南昌大學(xué);2014年

，

本文編號：1588107