本文選題：前列腺癌　切入點(diǎn)：IAP　出處：《天津醫(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BIRC6基因在前列腺癌中的表達(dá)水平及對前列腺癌細(xì)胞體內(nèi)、外生物學(xué)行為的影響,并觀察聯(lián)合降低以BIRC6為基礎(chǔ)的IAP基因家族表達(dá)對前列腺癌細(xì)胞生物學(xué)行為的影響。方法:運(yùn)用Western Blot與RT-qPCR檢測BIRC6在正常前列腺細(xì)胞RWPE-1與前列腺癌細(xì)胞系LNCaP、PC-3、C4-2和DU145中蛋白及mRNA的表達(dá)水平;構(gòu)建BIRC6低表達(dá)質(zhì)粒,擴(kuò)增后提取BIRC6低表達(dá)質(zhì)粒,轉(zhuǎn)染至PC-3細(xì)胞,分別應(yīng)用Western Blot與RT-qPCR驗(yàn)證BIRC6在PC-3細(xì)胞系中蛋白和mRNA表達(dá)水平;使用MTT、CCK-8方法檢測低表達(dá)BIRC6的PC-3的細(xì)胞增殖能力;應(yīng)用單克隆形成實(shí)驗(yàn)觀察BIRC6低表達(dá)后對PC-3細(xì)胞生長狀況的影響;用Transwell方法檢測BIRC6低表達(dá)對前列腺癌細(xì)胞侵襲能力的變化;采取磷脂酰絲氨酸外翻法(Annexin V)檢測BIRC6低表達(dá)后對前列腺癌凋亡的改變;使用流式細(xì)胞術(shù)分析BIRC-6低表達(dá)后對細(xì)胞生長周期的變化;將PC-3細(xì)胞種植于NOD-SCID小鼠皮下,使用BIRC6特異反義寡核酸腹腔注射觀察對腫瘤生長的影響。同時設(shè)計除BIRC6外的IAP家族基因BIRC2低表達(dá)質(zhì)粒,與BIRC6低表達(dá)質(zhì)粒共轉(zhuǎn)染PC-3細(xì)胞,觀察其對上述生物學(xué)行為的影響。記錄、整理實(shí)驗(yàn)數(shù)據(jù)及圖像資料,需使用統(tǒng)計分析的實(shí)驗(yàn)結(jié)果均運(yùn)用SPSS軟件進(jìn)行統(tǒng)計學(xué)分析。結(jié)果:前列腺癌細(xì)胞系中BIRC-6的蛋白及mRNA表達(dá)情況普遍高于正常前列腺細(xì)胞,并且在LNCaP及PC-3細(xì)胞系表達(dá)量顯著升高;我們發(fā)現(xiàn)BIRC6基因在CRPC患者組織中表達(dá)明顯升高,故選用了雄激素非依賴性PC-3細(xì)胞作為研究對象;構(gòu)建BIRC6低表達(dá)質(zhì)粒轉(zhuǎn)染PC-3細(xì)胞后發(fā)現(xiàn),BIRC6低表達(dá)可顯著抑制PC-3細(xì)胞生長,降低其侵襲能力,促進(jìn)細(xì)胞的凋亡,影響細(xì)胞生長周期,使停滯于G2/M期的細(xì)胞明顯增多。在同時轉(zhuǎn)染BIRC6與BIRC2低表達(dá)質(zhì)粒的PC-3細(xì)胞中,我們同樣觀察到抑制前列腺癌細(xì)胞增殖和促進(jìn)凋亡的現(xiàn)象,并且其抑制作用更加強(qiáng)烈。在小鼠皮下成瘤實(shí)驗(yàn)中,同時抑制兩種IAP基因的反義寡核苷酸可更有效的抑制腫瘤的生長。結(jié)論:BIRC6表達(dá)改變可影響前列腺癌細(xì)胞生物學(xué)行為,并且同時降低以BIRC6為基礎(chǔ)的聯(lián)合IAP家族成員BIRC2可更有效的抑制前列腺癌在體內(nèi)、外生長。
[Abstract]:Objective: to study the expression level of BIRC6 gene in prostate cancer and its effect on the biological behavior of prostate cancer cells in vivo and in vitro. To observe the effect of combined reduction of IAP gene family expression based on BIRC6 on the biological behavior of prostate cancer cells. Methods: Western Blot and RT-qPCR were used to detect the expression of BIRC6 in normal prostatic cells RWPE-1 and prostate cancer cell lines LNCaPnPC-3C4-2 and DU145. The expression level of white and mRNA; The low expression plasmid of BIRC6 was constructed, the low expression plasmid of BIRC6 was extracted and transfected into PC-3 cells, the expression level of BIRC6 and mRNA in PC-3 cell line were verified by Western Blot and RT-qPCR, and the proliferation ability of PC-3 with low expression of BIRC6 was detected by MTT- CCK-8 method. The effect of low expression of BIRC6 on the growth of PC-3 cells was observed by monoclonal formation assay, and the change of invasion ability of prostate cancer cells by low expression of BIRC6 was detected by Transwell method. The changes of apoptosis in prostate cancer were detected by Annexin V) after low expression of BIRC6. Flow cytometry was used to analyze the changes of cell cycle after low expression of BIRC-6. PC-3 cells were implanted subcutaneously in NOD-SCID mice. The effect of BIRC6 specific antisense oligodeoxynucleic acid (BIRC6) on tumor growth was observed by intraperitoneal injection. The low expression plasmid of IAP family gene BIRC2 except BIRC6 was designed and co-transfected with BIRC6 low expression plasmid to observe its effect on the above biological behavior. The experimental data and image data were analyzed by SPSS software. Results: the expression of BIRC-6 protein and mRNA in prostate cancer cell line was generally higher than that in normal prostate cell line. The expression of BIRC6 gene was significantly increased in the tissues of CRPC patients, so androgen independent PC-3 cells were selected as the research objects. After transfection of PC-3 cells with BIRC6 low expression plasmid, it was found that low expression of BIRC6 could significantly inhibit the growth of PC-3 cells, reduce its invasion ability, promote cell apoptosis and affect cell growth cycle. In PC-3 cells transfected with both BIRC6 and BIRC2 low expression plasmids, we also observed the inhibition of prostate cancer cell proliferation and the promotion of apoptosis. In murine subcutaneous tumorigenesis, antisense oligonucleotides of two IAP genes can inhibit tumor growth more effectively. Conclusion the change of the expression of BIRC6 may affect the biological behavior of prostate cancer cells. At the same time, the reduction of BIRC2, a member of the IAP family based on BIRC6, was more effective in inhibiting prostate cancer growth in vivo and in vitro.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.25

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