【摘要】：[目的]：以前列腺癌細(xì)胞LNCaP、PC3、DU145為研究對(duì)象,并建立前列腺癌細(xì)胞小鼠荷瘤模型,檢測(cè)KLF9基因在前列腺癌細(xì)胞系中的表達(dá)情況及其對(duì)前列腺癌細(xì)胞生物學(xué)效應(yīng)的影響,并初步探討其影響前列腺癌發(fā)生發(fā)展的可能機(jī)制。[方法]：在研究中,我們用實(shí)時(shí)熒光定量PCR(RT-PCR)和蛋白質(zhì)印跡法(Western Blot)檢測(cè)KLF9基因在抗雄藥氟他胺(flutamide)在去雄誘導(dǎo)LNCaP細(xì)胞凋亡過程中的變化,以及KLF9在不同前列腺(癌)細(xì)胞系中的表達(dá)情況。我們構(gòu)建了攜帶有四環(huán)素可調(diào)控(tetracycline-on, Tet-on)基因表達(dá)系統(tǒng)的真核表達(dá)載體pTRIPZ-KLF9,用慢病毒感染的方法導(dǎo)入人前列腺癌細(xì)胞系PC3和DU145中,并在強(qiáng)力霉素(doxycycline, Dox)的誘導(dǎo)下過表達(dá)KLF9,分別采用MTT法、克隆形成實(shí)驗(yàn)、FACS法、TUNEL染色及免疫組化(IHC)檢測(cè)KLF9過表達(dá)對(duì)前列腺癌細(xì)胞增殖、凋亡和細(xì)胞周期的影響；用構(gòu)建的pTRIPZ-KLF9和陰性對(duì)照質(zhì)粒轉(zhuǎn)染的PC3或LNCaP細(xì)胞接種于小鼠皮下,觀察轉(zhuǎn)染了各質(zhì)粒的PC3或LNCaP細(xì)胞在小鼠體內(nèi)成瘤率、瘤體生長(zhǎng)速度以及平均腫瘤體積等方面的變化。并且用Western Blot檢測(cè)了凋亡相關(guān)蛋白的變化情況以及可能的機(jī)制的變化情況,并對(duì)涉及機(jī)制的可能蛋白做了恢復(fù)實(shí)驗(yàn)。[結(jié)果]：在氟他胺抗雄誘導(dǎo)LNCaP細(xì)胞發(fā)生凋亡的過程中,可誘導(dǎo)KLF9的表達(dá),且具有隨時(shí)間變化的遞增關(guān)系;敲除內(nèi)源性的KLF9基因可以部分降低氟他胺對(duì)LNCaP細(xì)胞造成的生長(zhǎng)抑制。同時(shí)發(fā)現(xiàn),與非腫瘤細(xì)胞(RWPE-1、BPH1)相比,KLF9在前列腺癌細(xì)胞系中是低表達(dá)的,尤其是在惡性程度更高的雄激素非依賴性細(xì)胞系(PC3、DU145、C4-2B)中表達(dá)水平更低。過表達(dá)KLF9能顯著抑制前列腺癌PC3和DU145細(xì)胞的增殖及克隆形成能力(P0.001),并能明顯的誘導(dǎo)細(xì)胞凋亡(P0.001);且使細(xì)胞周期被阻滯在G 2/M期。在KLF9過表達(dá)抑制細(xì)胞增殖并且誘導(dǎo)凋亡的過程中,Western Blot及IHC實(shí)驗(yàn)結(jié)果顯示,抗凋亡相關(guān)蛋白BCL2、Bcl-xl的表達(dá)水平降低(P0.01),凋亡相關(guān)蛋白Cleaved Casspase3、Cleaved PARP的表達(dá)水平明顯升高。在體內(nèi)實(shí)驗(yàn)中,接種pTRIPZ-KLF9組的小鼠腫瘤成瘤率下降(P0.01),小鼠瘤體生長(zhǎng)速度明顯慢于對(duì)照組(P0.001),實(shí)驗(yàn)終止時(shí),腫瘤平均體積明顯小于對(duì)照組(P0.001)。在機(jī)制方面,KLF9過表達(dá)可抑制AKT的活化,同時(shí)也抑制AKT下游蛋白mTOR的活化;AKT過表達(dá)之后,可以部分恢復(fù)由KLF9誘導(dǎo)引起的對(duì)前列腺癌細(xì)胞PC3、DU145的生長(zhǎng)抑制及細(xì)胞凋亡。[結(jié)論]：1)轉(zhuǎn)錄因子KLF9在前列腺癌細(xì)胞系中的表達(dá)低于其在相應(yīng)的非腫瘤細(xì)胞系中的表達(dá)量；2)在抗雄藥物氟他胺誘導(dǎo)LNCaP細(xì)胞發(fā)生凋亡的過程中,KLF9基因的表達(dá)上調(diào)；3)在體外及體內(nèi)實(shí)驗(yàn)中,KLF9基因過表達(dá)后可顯著地誘導(dǎo)前列腺癌細(xì)胞發(fā)生凋亡,抑制前列腺癌的生長(zhǎng)及體內(nèi)成瘤；其可能為前列腺癌發(fā)病的一個(gè)抑癌基因;4)AKT信號(hào)通路的抑制參與了轉(zhuǎn)錄因子KLF9抑制前列腺癌細(xì)胞生長(zhǎng)的過程。
[Abstract]:[objective]: to investigate the expression of KLF9 gene in prostate cancer cell line LNCaP,PC3,DU145 and its effect on the biological effect of prostate cancer cell line. The possible mechanism of its influence on the occurrence and development of prostate cancer was discussed. [methods]: in our study, we used real-time fluorescent quantitative PCR (RT-PCR) and Western blot (Western Blot) to detect the changes of KLF9 gene in the process of deandrogen-induced LNCaP cell apoptosis induced by androgenic flutamide (flutamide). And the expression of KLF9 in different prostate (cancer) cell lines. We constructed an eukaryotic expression vector pTRIPZ-KLF9, carrying tetracycline regulated (tetracycline-on, Tet-on) gene expression system into human prostate cancer cell lines PC3 and DU145 by lentiviral infection, and overexpressed KLF9, by MTT method under the induction of doxycycline (doxycycline, Dox). The effects of overexpression of KLF9 on the proliferation, apoptosis and cell cycle of prostate cancer cells were detected by FACS assay, TUNEL staining and immunohistochemical (IHC). The PC3 or LNCaP cells transfected with constructed pTRIPZ-KLF9 and negative control plasmids were inoculated subcutaneously in mice. The tumorigenic rate, tumor growth rate and mean tumor volume of PC3 or LNCaP cells transfected with each plasmid in mice were observed. Western Blot was used to detect the changes of apoptosis-related proteins and possible mechanisms. [results]: in the course of anti-androgen-induced apoptosis of LNCaP cells, flutamide could induce the expression of KLF9 and increase with time, and knockout of endogenous KLF9 gene could partly reduce the growth inhibition of LNCaP cells induced by flutamide. It was also found that KLF9 expression was lower in prostate cancer cell lines than in non-tumor cells (RWPE-1,BPH1), especially in androgen independent cell lines (PC3,DU145,C4-2B) with higher malignancy. Overexpression of KLF9 could significantly inhibit the proliferation and clone formation of prostate cancer PC3 and DU145 cells (P0.001), induce apoptosis (P0.001), and block the cell cycle in G _ 2 / M phase. The results of, Western Blot and IHC experiments showed that the expression of anti-apoptosis-related protein (BCL2,Bcl-xl) was decreased (P0.01) and the expression of apoptosis-related protein (Cleaved Casspase3,Cleaved PARP) was significantly increased during the process of over-expression of KLF9 inhibiting cell proliferation and inducing apoptosis. In vivo, the tumorigenesis rate of mice inoculated with pTRIPZ-KLF9 decreased (P0.01), and the tumor growth rate of mice was significantly slower than that of control group (P0.001). At the end of the experiment, the average tumor volume was significantly smaller than that of the control group (P0.001). In terms of mechanism, overexpression of KLF9 could inhibit the activation of AKT and the activation of mTOR, the downstream protein of AKT, and AKT overexpression could partially restore the growth inhibition and apoptosis of prostate cancer cell line PC3,DU145 induced by KLF9. [conclusion]: 1) the expression of transcription factor KLF9 in prostate cancer cell line is lower than that in the corresponding non-tumor cell line, 2) the expression of KLF9 gene is up-regulated during the apoptosis of LNCaP cells induced by androgenic flutamide. 3) in vitro and in vivo experiments, overexpression of KLF9 gene could significantly induce apoptosis of prostate cancer cells, inhibit the growth of prostate cancer and tumorigenesis in vivo. It may be a tumor suppressor gene in the pathogenesis of prostate cancer. 4) the inhibition of AKT signaling pathway is involved in the inhibition of the growth of prostate cancer cells by the transcription factor KLF9.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.25

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