本文選題：膀胱癌 + RNA干擾�。� 參考：《桂林醫(yī)學(xué)院》2015年碩士論文

【摘要】：目的:Livin是IAP家族的最新成員。其在正常膀胱組織中不表達,而在人膀胱癌細胞中的高表達可刺激腫瘤細胞的增殖,并提高膀胱癌的術(shù)后復(fù)發(fā)率,更能使腫瘤細胞對化療藥物產(chǎn)生耐藥性。因此,其可成為膀胱癌的治療靶點及預(yù)后復(fù)發(fā)的監(jiān)測指標(biāo)。本研究通過觀察siRNA抑制人膀胱癌EJ細胞中Livin基因的表達對細胞增殖、凋亡及化療敏感性的影響,為膀胱癌發(fā)病機制的研究,以及對膀胱癌的基因和化學(xué)治療提供理論基礎(chǔ)和實驗依據(jù)。方法:(1)觀察通過不同條件轉(zhuǎn)染過的EJ細胞,進行siRNA轉(zhuǎn)染細胞條件的優(yōu)化及效率的評價。(2)采用脂質(zhì)體轉(zhuǎn)染技術(shù)將人工合成的特異性Livin siRNA轉(zhuǎn)入人膀胱癌EJ細胞中。(3)經(jīng)Livin siRNA轉(zhuǎn)染細胞24h后,半定量RT-PCR檢測各組EJ細胞中Livin mRNA的表達。(4)Western Blot進一步驗證轉(zhuǎn)染48h后細胞中Livin蛋白的表達。(5)選用THP作用于轉(zhuǎn)染和未轉(zhuǎn)染的細胞,CCK-8法檢測各組細胞的增殖抑制率。(6)流式細胞術(shù)檢測各組細胞的凋亡情況。結(jié)果:(1)用FAM標(biāo)記的Negative control siRNA轉(zhuǎn)染6孔板中的人膀胱癌EJ細胞,觀察到當(dāng)細胞數(shù)量為3×105/孔,si RNA為100pmol/孔、Lipofectamine#174;2000試劑為5μl/孔時,有較高轉(zhuǎn)染效率(70%)。當(dāng)細胞密度為4×103/孔接種于96孔板中,siRNA為5pmol/孔、Lipofectamine#174;2000試劑為0.25μl/孔時,有較高轉(zhuǎn)染效率(70%);(2)Livin特異性siRNA轉(zhuǎn)染人膀胱癌EJ細胞24h后,干擾組(轉(zhuǎn)染siRNA)中Livin mRNA的表達較空白對照組及陰性對照組顯著降低(P0.05);(3)轉(zhuǎn)染48h后,Livin在蛋白水平的表達,較空白對照組及陰性對照組明顯下調(diào)(P0.05);(4)經(jīng)CCK-8檢測發(fā)現(xiàn),Livin siRNA干擾組、THP化療組及干擾+化療的聯(lián)合作用組均可抑制EJ細胞的增殖。且聯(lián)合作用組對細胞的增殖抑制率顯著高于單獨干擾組及單獨化療作用組(P0.05);(5)流式細胞術(shù)的檢測結(jié)果顯示,與空白對照組比較,干擾組、化療組及聯(lián)合作用組均可促使膀胱癌EJ細胞的凋亡,且聯(lián)合作用組的細胞凋亡率顯著高于單獨干擾組及單獨化療作用組(P0.05)。結(jié)論:(1)Livin基因在膀胱癌的發(fā)生發(fā)展過程中起到一定的促進作用;(2)本實驗設(shè)計的Livin特異性siRNA能夠有效抑制人膀胱癌EJ細胞中Livin基因的表達;(3)通過抑制Livin基因的表達,可有效抑制人膀胱癌EJ細胞的增殖、并促進其凋亡;(4)經(jīng)Livin siRNA轉(zhuǎn)染的EJ細胞加強了對化療藥物THP的敏感性。
[Abstract]:Objective: Livin is the newest member of the IAP family.It is not expressed in normal bladder tissue, but high expression in human bladder cancer cells can stimulate the proliferation of tumor cells, increase the recurrence rate of bladder cancer, and make the tumor cells become more resistant to chemotherapeutic drugs.Therefore, it can be used as a therapeutic target and a predictor of recurrence of bladder cancer.In this study, the effects of siRNA on the proliferation, apoptosis and chemosensitivity of human bladder cancer EJ cells were studied.And to provide theoretical and experimental basis for gene and chemotherapy of bladder cancer.Methods the EJ cells transfected with different conditions were observed.Optimization of siRNA transfection conditions and evaluation of its efficiency. 2) transfection of synthetic specific Livin siRNA into human bladder cancer EJ cells by liposome transfection with Livin siRNA for 24 h.Semi-quantitative RT-PCR Detection of Livin mRNA expression in EJ cells. Further verify the expression of Livin protein in EJ cells 48 h after transfection. Use THP on transfected and untransfected EJ cells to detect the inhibitory rate of proliferation of EJ cells in each group by CCK-8 method.The apoptosis of the cells in each group was detected by cytology.When the cell density was 4 脳 10 3 / well inoculated in 96 well plate, the siRNA was 5pmol/ Hole Lipofectamine #174A reagent was 0.25 渭 l / well, the transfection efficiency was higher than 70% Livin specific siRNA transfection to human bladder cancer EJ cells for 24 h.The expression of Livin mRNA in the interference group (transfected siRNAs) was significantly lower than that in the blank control group and the negative control group.Compared with the blank control group and the negative control group, the P0. 05 P0. 05 and P0. 05 were significantly down-regulated by CCK-8. The results of CCK-8 test showed that the proliferation of EJ cells could be inhibited in both the THP chemotherapy group and the combined treatment group.The inhibition rate of cell proliferation in the combined group was significantly higher than that in the control group and the chemotherapy alone group. The results of flow cytometry showed that compared with the blank control group, the inhibition rate of the cell proliferation in the combined group was significantly higher than that in the control group.The apoptosis rate of EJ cells in combination group and chemotherapy group was significantly higher than that in control group and chemotherapy alone group (P 0.05).Conclusion Livin specific siRNA can effectively inhibit the expression of Livin gene in human bladder cancer EJ cells by inhibiting the expression of Livin gene.EJ cells transfected with Livin siRNA enhanced the sensitivity to the chemotherapeutic drug THP.
【學(xué)位授予單位】：桂林醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R737.14

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