本文選題：E2F1 + TFDP3��； 參考：《第四軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：前列腺癌(prostate cancer,PCa)是男性腫瘤中僅次于肺癌的惡性腫瘤之一[1]。但是由于前列腺癌大多發(fā)生隱匿,一旦確診,很大一部分患者基本已經(jīng)到了中晚期,預(yù)后較差。目前治療前列腺癌的一般方式是去雄激素治療,旨在激素治療后誘導(dǎo)腫瘤細(xì)胞的凋亡。但是這種治療措施在初期雖然有效,一段時(shí)間后幾乎所有患者都會(huì)由雄激素依賴轉(zhuǎn)變?yōu)榉切奂に匾蕾囆?最終導(dǎo)致腫瘤的進(jìn)展或轉(zhuǎn)移。雄激素依賴到非依賴的轉(zhuǎn)變機(jī)制在各領(lǐng)域研究備受關(guān)注,目前已知的機(jī)制包括雄激素受體(AR)突變、克隆選擇、共激活因子(Coactivator)的表達(dá)等,目的都是各種方式避免前列腺癌細(xì)胞的凋亡。研究表明,自噬是降解細(xì)胞器的主要途徑,一定條件下,在細(xì)胞凋亡中自噬可能會(huì)起到保護(hù)作用,抑制細(xì)胞自噬會(huì)誘導(dǎo)細(xì)胞的凋亡[2]。另一方面,細(xì)胞的凋亡途徑受到抑制后也可能導(dǎo)致細(xì)胞的自噬性死亡[3]。目前,在前列腺中雄激素非依賴的形成是否和自噬有一定的調(diào)控關(guān)系,或者在前列腺中自噬和凋亡如何被調(diào)控,并沒有明確的結(jié)論。TFDP3是新發(fā)現(xiàn)的E2F1的直接調(diào)控因子。有研究表明TFDP3不僅可以抑制E2F1誘導(dǎo)的細(xì)胞增殖,而且還可以抑制E2F1誘導(dǎo)的細(xì)胞凋亡;但是在前列腺癌中是否參與了凋亡和自噬作用有待研究及進(jìn)一步驗(yàn)證。本研究通過免疫組化和原位雜交探索TFDP3在前列腺癌組織中的表達(dá);通過半定量RT-PCR檢測(cè)TFDP3在前列腺癌細(xì)胞中的表達(dá)。然后,分別通過RT-PCR和Western blot初步探索TFDP3在E2F1作用后自噬相關(guān)蛋白LC3B基因水平和蛋白水平的變化,并且利用流式細(xì)胞儀檢測(cè)TFDP3與E2F1的相互作用對(duì)前列腺癌細(xì)胞凋亡的影響。主要研究?jī)?nèi)容包括:1..通過NCBI基因組數(shù)據(jù)庫(kù)檢索到TFDP3基因的DNA序列,利用軟件Primer5.0設(shè)計(jì)引物。以前列腺癌LNCaP細(xì)胞基因組DNA作為模板,采用PCR技術(shù)擴(kuò)增TFDP3片段,長(zhǎng)約1244bp。RT-PCR,原位雜交雜交和免疫組化結(jié)果表明,TFDP3在前列腺癌細(xì)胞和組織中均有不同程度的表達(dá)。2.通過轉(zhuǎn)染pcDNA3.1-TFDP3和PCMV-E2F1-HA于LNCaP細(xì)胞后,RT-PCR和Western blot檢測(cè)結(jié)果均顯示,TFDP3高表達(dá)的LNCaP細(xì)胞中自噬相關(guān)基因LC3B表達(dá)水平明顯升高;而轉(zhuǎn)染PCMV-E2F1-HA后LNCaP細(xì)胞中LC3B水平明顯降低(P0.05)。表明轉(zhuǎn)錄因子TFDP3過表達(dá)可以誘導(dǎo)LNCaP細(xì)胞的自噬現(xiàn)象,同時(shí)提示E2F1和TFDP3的相互作用可以抑制LNCaP細(xì)胞的自噬功能。3.通過流式細(xì)胞儀檢測(cè)TFDP3與E2F1相互作用后前列腺癌細(xì)胞凋亡的變化,可以發(fā)現(xiàn):PCMV-E2F1-HA重組質(zhì)粒轉(zhuǎn)染后細(xì)胞凋亡率明顯上升(P0.05),而與pcDNA3.1-TFDP3共轉(zhuǎn)染后細(xì)胞凋亡率明顯下降(P0.05)。證實(shí)TFDP3可以抑制E2F1誘導(dǎo)的細(xì)胞凋亡。以上研究我們可以得到如下結(jié)論,TFDP3可以誘導(dǎo)LNCaP細(xì)胞中自噬基因LC3B的表達(dá),同時(shí)抑制E2F1誘導(dǎo)的細(xì)胞凋亡,提示TFDP3誘導(dǎo)的自噬功能和抗凋亡效應(yīng)極有能是前列腺癌由激素依賴性向非依賴性轉(zhuǎn)變過程中的關(guān)鍵的自我保護(hù)機(jī)制之一。該研究的結(jié)果也為TFDP3是否能作為前列腺癌治療的靶點(diǎn)治療提供了新的研究基礎(chǔ)。
[Abstract]:Prostate cancer (PCa) is one of the malignant tumors that are second only to lung cancer in male tumor [1]., but because most of the prostate cancer is occult, once the diagnosis is confirmed, a large part of the patients are basically in the middle and late stages, and the prognosis is poor. The general way to treat prostate cancer is to go to androgen therapy, which is designed to induce hormone therapy. However, although this treatment is effective in the early stages, almost all patients change from androgen dependence to non androgen dependence after a period of time, and eventually lead to the progression or metastasis of the tumor. Androgen dependence on the non dependent transformation mechanism has attracted much attention in all fields, and the present mechanism includes the male irritable mechanism. AR mutation, clone selection, the expression of CO activation factor (Coactivator) and so on. The aim is to avoid the apoptosis of prostate cancer cells in various ways. The study shows that autophagy is the main way to degrade the organelles. Under certain conditions, autophagy may play a protective role in cell apoptosis, and inhibition of autophagy induces cell apoptosis, [2]. On the other hand, the inhibition of cell apoptosis may also lead to the autophagic death of the cell [3].. Whether the androgen independent formation in the prostate is regulated by autophagy, or how the autophagy and apoptosis in the prostate is regulated, and the.TFDP3 is a direct regulation of the newly discovered E2F1. Factors. Some studies have shown that TFDP3 not only inhibits the proliferation of E2F1 induced cells, but also inhibits apoptosis induced by E2F1, but whether it is involved in apoptosis and autophagy in prostate cancer remains to be studied and further verified. This study explored the expression of TFDP3 in prostate cancer by immunohistochemistry and in situ hybridization; The expression of TFDP3 in prostate cancer cells was detected by semi quantitative RT-PCR. Then, the changes in the level and protein level of the autophagic related protein LC3B gene were preliminarily explored by RT-PCR and Western blot respectively, and the effect of the interaction between TFDP3 and E2F1 on the apoptosis of prostate cancer cells was detected by flow cytometry. The research contents include: 1.. Retrieves the DNA sequence of the TFDP3 gene through the NCBI genome database and uses the software Primer5.0 to design primers. The DNA of the prostate cancer LNCaP cell genome DNA is used as a template, and PCR technology is used to amplify the TFDP3 fragment, long approximately 1244bp.RT-PCR, in situ hybridization and immunization histochemical results show that TFDP3 is in the prostate cancer cells and groups. After transfection of pcDNA3.1-TFDP3 and PCMV-E2F1-HA to LNCaP cells, the results of RT-PCR and Western blot detected by.2. showed that the expression level of autophagy related gene LC3B expressed in LNCaP cells with high expression of TFDP3 obviously increased, and the level in the LNCaP fine cells decreased significantly after the transfection. The overexpression of TFDP3 can induce autophagy in LNCaP cells, and the interaction of E2F1 and TFDP3 can inhibit the autophagy function of LNCaP cells.3. by flow cytometry to detect the apoptosis of prostate cancer cells after the interaction between TFDP3 and E2F1. It is found that the apoptosis rate of the recombinant plasmid is obviously increased after the recombinant plasmid is transfected (P). 0.05), the apoptosis rate of the cells decreased significantly after CO transfection with pcDNA3.1-TFDP3 (P0.05). It was proved that TFDP3 could inhibit the apoptosis induced by E2F1. We can conclude that TFDP3 can induce the expression of autophagic gene LC3B in LNCaP cells and inhibit the apoptosis induced by E2F1, suggesting the function and resistance of TFDP3 induced autophagy. The effect of apoptosis is one of the key self-protection mechanisms of prostate cancer in the process of hormone dependence to non dependence. The results of this study also provide a new basis for TFDP3 as a target therapy for prostate cancer treatment.
【學(xué)位授予單位】：第四軍醫(yī)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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相關(guān)碩士學(xué)位論文 前2條

1 任麗芬;TFDP3對(duì)前列腺癌LNCaP細(xì)胞自噬及凋亡調(diào)控的初步研究[D];第四軍醫(yī)大學(xué);2014年

2 李蕊;TFDP3的表達(dá)調(diào)控及其對(duì)前列腺癌PC3細(xì)胞凋亡的影響[D];第四軍醫(yī)大學(xué);2014年

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本文編號(hào)：2081140