TFDP3对前列腺癌LNCaP细胞自噬及凋亡调控的初步研究

发布时间：2018-06-29 06:02

本文选题：E2F1 + TFDP3　；参考：《第四军医大学》2014年硕士论文

【摘要】：前列腺癌(prostate cancer,PCa)是男性肿瘤中仅次于肺癌的恶性肿瘤之一[1]。但是由于前列腺癌大多发生隐匿,一旦确诊,很大一部分患者基本已经到了中晚期,预后较差。目前治疗前列腺癌的一般方式是去雄激素治疗,旨在激素治疗后诱导肿瘤细胞的凋亡。但是这种治疗措施在初期虽然有效,一段时间后几乎所有患者都会由雄激素依赖转变为非雄激素依赖性,最终导致肿瘤的进展或转移。雄激素依赖到非依赖的转变机制在各领域研究备受关注,目前已知的机制包括雄激素受体(AR)突变、克隆选择、共激活因子(Coactivator)的表达等,目的都是各种方式避免前列腺癌细胞的凋亡。研究表明,自噬是降解细胞器的主要途径,一定条件下,在细胞凋亡中自噬可能会起到保护作用,抑制细胞自噬会诱导细胞的凋亡[2]。另一方面,细胞的凋亡途径受到抑制后也可能导致细胞的自噬性死亡[3]。目前,在前列腺中雄激素非依赖的形成是否和自噬有一定的调控关系,或者在前列腺中自噬和凋亡如何被调控,并没有明确的结论。TFDP3是新发现的E2F1的直接调控因子。有研究表明TFDP3不仅可以抑制E2F1诱导的细胞增殖,而且还可以抑制E2F1诱导的细胞凋亡;但是在前列腺癌中是否参与了凋亡和自噬作用有待研究及进一步验证。本研究通过免疫组化和原位杂交探索TFDP3在前列腺癌组织中的表达;通过半定量RT-PCR检测TFDP3在前列腺癌细胞中的表达。然后,分别通过RT-PCR和Western blot初步探索TFDP3在E2F1作用后自噬相关蛋白LC3B基因水平和蛋白水平的变化,并且利用流式细胞仪检测TFDP3与E2F1的相互作用对前列腺癌细胞凋亡的影响。主要研究内容包括:1..通过NCBI基因组数据库检索到TFDP3基因的DNA序列,利用软件Primer5.0设计引物。以前列腺癌LNCaP细胞基因组DNA作为模板,采用PCR技术扩增TFDP3片段,长约1244bp。RT-PCR,原位杂交杂交和免疫组化结果表明,TFDP3在前列腺癌细胞和组织中均有不同程度的表达。2.通过转染pcDNA3.1-TFDP3和PCMV-E2F1-HA于LNCaP细胞后,RT-PCR和Western blot检测结果均显示,TFDP3高表达的LNCaP细胞中自噬相关基因LC3B表达水平明显升高;而转染PCMV-E2F1-HA后LNCaP细胞中LC3B水平明显降低(P0.05)。表明转录因子TFDP3过表达可以诱导LNCaP细胞的自噬现象,同时提示E2F1和TFDP3的相互作用可以抑制LNCaP细胞的自噬功能。3.通过流式细胞仪检测TFDP3与E2F1相互作用后前列腺癌细胞凋亡的变化,可以发现:PCMV-E2F1-HA重组质粒转染后细胞凋亡率明显上升(P0.05),而与pcDNA3.1-TFDP3共转染后细胞凋亡率明显下降(P0.05)。证实TFDP3可以抑制E2F1诱导的细胞凋亡。以上研究我们可以得到如下结论,TFDP3可以诱导LNCaP细胞中自噬基因LC3B的表达,同时抑制E2F1诱导的细胞凋亡,提示TFDP3诱导的自噬功能和抗凋亡效应极有能是前列腺癌由激素依赖性向非依赖性转变过程中的关键的自我保护机制之一。该研究的结果也为TFDP3是否能作为前列腺癌治疗的靶点治疗提供了新的研究基础。
[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.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25

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