本文選題：前列腺癌 + VPA��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：研究背景前列腺癌是全球范圍內(nèi)男性第二大惡性腫瘤,我國前列腺癌的發(fā)病率近十年來逐年增長(zhǎng)。在我國,超過40%的前列腺癌患者確診時(shí)已經(jīng)進(jìn)入中晚期,遠(yuǎn)遠(yuǎn)高于歐美水平。雄激素剝奪治療(Androgen deprivation therapy,ADT)是國內(nèi)外晚期前列腺癌的主要治療方式,但其有效期一般僅能維持18-24個(gè)月。在ADT治療失效之后,疾病將發(fā)展為去勢(shì)抵抗性前列腺癌(Castrateresistant prostate cancer,CRPC),部分患者的腫瘤甚至進(jìn)一步轉(zhuǎn)移至前列腺以外的其它器官。針對(duì)轉(zhuǎn)移性去勢(shì)抵抗性前列腺癌雖然可以選擇新型雄激素生物合成酶抑制劑治療,但作用有限。因此,尋求更多更有效的治療方法是目前研究的方向。上皮間充質(zhì)轉(zhuǎn)化(Epithelial-mesenchymal transition,EMT)在腫瘤的發(fā)生發(fā)展過程中起到重要的調(diào)控作用,在腫瘤細(xì)胞的EMT過程中,上皮細(xì)胞逐漸失去了本身的特性,侵入上皮基底膜并通過進(jìn)入血液循環(huán)系統(tǒng)獲得了浸潤周圍組織或遠(yuǎn)處轉(zhuǎn)移的能力。除了失去胞間鏈接外,發(fā)生EMT的細(xì)胞逐漸失去了其細(xì)胞極性,上皮標(biāo)記物諸如E鈣黏蛋白減少,而N鈣黏蛋白等間充質(zhì)細(xì)胞標(biāo)記物逐漸增多,因此,具有此種特性的細(xì)胞更易向鄰近組織浸潤性生長(zhǎng)或發(fā)生遠(yuǎn)處轉(zhuǎn)移。腫瘤相關(guān)的EMT并不是單一的過程,腫瘤細(xì)胞除獲得了遷移和侵襲能力外,還具備了復(fù)雜且全面地參與表觀遺傳重組、新陳代謝和分化等能力。在前列腺癌病人腫瘤進(jìn)展過程中,EMT在前列腺癌由原位細(xì)胞向轉(zhuǎn)移性細(xì)胞的轉(zhuǎn)化發(fā)展過程中起到了非常重要的作用,此類前列腺癌細(xì)胞失去了本身的上皮特性并獲得了間充質(zhì)細(xì)胞的特性。EMT同樣也促進(jìn)了去勢(shì)抵抗性前列腺癌的發(fā)展過程。EMT過程受體內(nèi)多個(gè)信號(hào)通路的調(diào)節(jié),其中轉(zhuǎn)化生長(zhǎng)因子β(Transforming growth factor β,TGF-β)信號(hào)通路起主要作用,SMAD4 蛋白作為TGF-β信號(hào)通路信號(hào)傳遞過程中重要的載體蛋白,被認(rèn)為是前列腺癌的促癌因子。E3 泛素連接酶-轉(zhuǎn)錄中介因子 1γ(Transcriptional intermediary factor1 γ,TIF1 γ)被認(rèn)為通過單泛素化修飾SMAD4蛋白,促使其與磷酸化的SMAD3蛋白解離,參與了 TGF-β/SMAD蛋白信號(hào)系統(tǒng)的調(diào)節(jié)過程。本課題組的前期相關(guān)研究顯示,丙戊酸鈉(Valproic acid,VPA)通過抑制SMAD4蛋白的表達(dá)逆轉(zhuǎn)了前列腺癌EMT過程。在本課題中,我們收集了山東省立醫(yī)院泌尿微創(chuàng)中心2012年以來收治的20例接受腹腔鏡前列腺癌根治術(shù)患者的前列腺癌組織及癌旁組織,應(yīng)用免疫組化技術(shù)分析SMAD4及TIF1 γ蛋白在前列腺癌患者與正常人之間的不同表達(dá)水平以及各蛋白與臨床病理變量的關(guān)系;體外實(shí)驗(yàn)部分,我們運(yùn)用了免疫組化、免疫共沉淀、免疫印跡等技術(shù),證實(shí)了 TIF1γ對(duì)SMAD4蛋白的單泛素化修飾作用是介導(dǎo)VPA抑制前列腺癌EMT過程的具體分子機(jī)制。研究目的1.應(yīng)用生物信息學(xué)分析前列腺癌相關(guān)蛋白在前列腺癌患者與正常人之間的不同表達(dá)水平以及各蛋白與臨床病理變量的關(guān)系。2.制作20例前列腺癌組織及其癌旁組織標(biāo)本的石蠟切片,應(yīng)用免疫組化技術(shù)檢測(cè)SMAD4及TIF1 γ蛋白的表達(dá)情況。3.體內(nèi)、外實(shí)驗(yàn)部分,分別培養(yǎng)PC3細(xì)胞及LNCaP細(xì)胞,構(gòu)建荷瘤小鼠模型,并用VPA處理,檢測(cè)TIF1 γ、SMAD4及EMT過程中相關(guān)標(biāo)記蛋白在不同處理組的表達(dá)量,探討VPA抑制前列腺癌EMT過程的具體機(jī)制。研究方法1.應(yīng)用石蠟包埋切片技術(shù)處理臨床采集的20例前列腺癌患者的前列腺癌組織及癌旁組織標(biāo)本,應(yīng)用免疫組化技術(shù)檢測(cè)TIF1γ、SMAD4蛋白在不同組織標(biāo)本中表達(dá)量的差異,結(jié)合患者隨訪資料,應(yīng)用統(tǒng)計(jì)學(xué)方法分析目的蛋白在前列腺癌患者與正常人之間不同表達(dá)水平以及各蛋白與臨床病理變量的關(guān)系,探討目的蛋白的表達(dá)與患者預(yù)后的關(guān)系,以期尋找新的前列腺癌預(yù)后指標(biāo)和潛在的治療靶點(diǎn)。2.采用免疫印跡、免疫共沉淀及免疫組化等分子生物學(xué)技術(shù)檢測(cè)前列腺癌細(xì)胞及荷瘤小鼠瘤體在VPA處理組與對(duì)照組中相關(guān)蛋白的表達(dá)情況,探討VPA調(diào)控TIF1 γ抑制前列腺癌細(xì)胞EMT過程及其與單泛素化SMAD4蛋白之間的關(guān)系。研究結(jié)果1.VPA升高了野生型PC3/LNCaP細(xì)胞內(nèi)單泛素化SMAD4蛋白水平,但無法影響SMAD4點(diǎn)突變的PC3/LNCaP細(xì)胞內(nèi)單泛素化SMAD4蛋白的水平。2.VPA通過調(diào)控TIF1 γ蛋白抑制前列腺癌細(xì)胞EMT過程。3.VPA抑制了荷瘤小鼠瘤體的生長(zhǎng)。4.前列腺癌組織內(nèi)TIF11γ與SMAD4蛋白含量呈負(fù)相關(guān),免疫組化結(jié)果顯示前列腺癌組織內(nèi)TIF1 γ含量明顯低于癌旁組織,SMAD4含量與上述結(jié)果相反。5.體內(nèi)實(shí)驗(yàn)結(jié)果顯示,相較于對(duì)照組,VPA處理組中TIF1γ表達(dá)量升高,而SMAD4蛋白表達(dá)量下降。研究結(jié)論VPA通過加速SMAD3/SMAD4復(fù)合物的核內(nèi)解離過程,降低核內(nèi)SMAD3/SMAD4復(fù)合物含量,從而抑制前列腺癌細(xì)胞EMT過程,TIF1γ對(duì)SMAD4的單泛素化修飾作用是VPA扣動(dòng)這一過程的"扳機(jī)"。研究意義本研究證實(shí)了 TIF1 γ介導(dǎo)VPA抑制前列腺癌EMT過程的具體分子機(jī)制,旨在證明VPA治療進(jìn)展期前列腺癌的可能性,豐富了進(jìn)展期前列腺癌的治療策略,并為臨床用藥提供了較為詳實(shí)的統(tǒng)計(jì)學(xué)依據(jù)。
[Abstract]:Background prostate cancer is the second major male malignant tumor around the world. The incidence of prostate cancer in China has increased year by year in the past ten years. In China, more than 40% of the prostate cancer patients have entered the middle and late stages, far higher than the European and American levels. Androgen deprivation therapy (Androgen deprivation therapy, ADT) is advanced at home and abroad. The main treatment of adenocarcinoma, but the duration of its validity is generally only 18-24 months. After the failure of ADT treatment, the disease will develop to Castrateresistant prostate cancer (CRPC), some of the patients may even be further transferred to other organs other than the prostate. Although adenocarcinoma can choose the new androgen biosynthesis inhibitor therapy, the effect is limited. Therefore, it is the current research direction to seek more and more effective treatment methods. Epithelial-mesenchymal transition (EMT) plays an important role in the development of tumor, and the EMT over the tumor cells is over. During the process, the epithelial cells gradually lose their own characteristics, invade the epithelial basement membrane and get the ability to infiltrate the surrounding tissues or distant metastases through the blood circulation system. In addition to the loss of intercellular links, EMT cells gradually lose their cell polarity, epithelial markers such as E calcium mucin and N calcium mucin. As a result, the cells with this characteristic are more susceptible to infiltrating or distant metastasis to adjacent tissues. The tumor related EMT is not a single process. In addition to the ability to migrate and invasiveness, the tumor cells have a complex and comprehensive participation in epigenetic reorganization, metabolism and differentiation. In the process of cancer progression in patients with prostate cancer, EMT plays a very important role in the transformation and development of prostate cancer from in situ cells to metastatic cells. This kind of prostate cancer cells lose their own epithelial properties and obtain the characteristics of mesenchymal cells.EMT, which also promote the development of castration resistance to prostate cancer. There are several signaling pathways within the.EMT process receptor, in which TGF beta (Transforming growth factor beta, TGF- beta) signaling pathway plays a major role, and SMAD4 protein is an important carrier protein in the signaling process of TGF- beta signaling. It is considered to be a cancer promoting factor.E3 ubiquitin ligase transcription mediator for Prost adenocarcinoma. Factor 1 gamma (Transcriptional intermediary factor1 gamma, TIF1 gamma) is considered to modify the SMAD4 protein by single ubiquitination to dissociate the phosphorylated SMAD3 protein and participate in the regulation process of the TGF- beta /SMAD protein signal system. Previous studies in this group showed that the sodium valproate (Valproic acid, VPA) could inhibit the SMAD4 protein. The expression reverses the EMT process of prostate cancer. In this subject, we collected 20 cases of prostate cancer tissue and para cancer tissue in patients with laparoscopic radical prostatectomy since 2012 in the urinary minimally invasive center of Shangdong Province-owned Hospital. The immunohistochemical technique was used to analyze the SMAD4 and TIF1 gamma protein in patients with prostate cancer and normal people. The relationship between different levels of expression and the relationship between the protein and the clinicopathological variables; in vitro experiment part, we used immunohistochemistry, immunoblotting and immunoblotting techniques to confirm that the single ubiquitination modification of TIF1 gamma to SMAD4 protein is a specific molecular mechanism to mediate the EMT process of prostate cancer by VPA. Objective 1. application of Biology Informatics analysis of the different expression levels of prostate cancer related proteins in the prostate cancer patients and normal people and the relationship between the protein and the clinicopathological variables.2. made paraffin sections of 20 cases of prostate cancer tissue and its paracancerous tissue specimens. The expression of SMAD4 and TIF1 gamma protein was detected by immunohistochemical technique in.3.. The PC3 cells and LNCaP cells were cultured, and the tumor bearing mice model was constructed, and the expression of related marker proteins in the process of TIF1 gamma, SMAD4 and EMT in different treatment groups was detected by VPA treatment. The specific mechanism of VPA inhibition of EMT process in prostate cancer was investigated. Method 1. the study method of paraffin embedded section was used to deal with the 20 cases of clinical collection. The differences in the expression of TIF1 gamma and SMAD4 protein in different tissue specimens were detected by immunohistochemical technique in the prostate cancer tissues and para cancer tissues of the patients with adenocarcinoma. The different expression levels of the target protein in the patients with prostate cancer and the normal people were analyzed by the statistical method, and the protein and Clinicopathology were analyzed by the statistical method. The relationship between the expression of the variable and the relationship between the expression of the target protein and the prognosis of the patient in order to find a new prognostic indicator and a potential therapeutic target for prostate cancer,.2. using immunoblotting, immunoblotting and immunohistochemistry to detect the related proteins in the VPA treatment group and the control group in the tumor bearing mice and the tumor bearing mice. The relationship between VPA regulation of TIF1 gamma inhibition of EMT process and the relationship with mono ubiquitin SMAD4 protein in prostate cancer cells was investigated. Results 1.VPA increased the level of ubiquitin SMAD4 protein in wild PC3/LNCaP cells, but could not affect the level of mono ubiquitin SMAD4 protein in PC3/LNCaP cells of SMAD4 point mutation. The inhibitory effect of TIF1 gamma protein on the EMT process of prostate cancer cell.3.VPA inhibits the growth of the tumor of tumor bearing mice and the TIF11 gamma content in the.4. prostate cancer tissue is negatively correlated. The immunohistochemical results show that the content of TIF1 gamma in the prostate cancer tissue is significantly lower than that in the para cancerous tissue, and the SMAD4 content is contrary to the results in the experimental results of the above results. Compared to the control group, the expression of TIF1 gamma in the VPA treatment group increased and the expression of SMAD4 protein decreased. Conclusion VPA can inhibit the EMT process in the prostate cancer cells by accelerating the intra nuclear dissociation process of SMAD3/SMAD4 complex and thus inhibiting the EMT process in the prostate cancer cells. The single ubiquitination modification of TIF1 gamma is the process of VPA deduction. The study confirms the specific molecular mechanism of TIF1 gamma mediated VPA inhibition of prostate cancer EMT process, which aims to prove the possibility of the treatment of advanced prostate cancer by VPA, enriching the treatment strategy of advanced prostate cancer and providing a more detailed statistical basis for clinical use of drugs.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.25

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