本文關(guān)鍵詞：氯�？朔雍退骼悄釁f(xié)同作用前列腺癌PC3細(xì)胞的機(jī)制研究以及miR-4293 rs12220909多態(tài)性與肺癌易感性的關(guān)聯(lián)分析　出處：《蘇州大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 氯�？朔� 索拉非尼 協(xié)同性 肺癌 miR-4293

【摘要】：前列腺癌是男性生殖系腫瘤中常見疾病之一,其發(fā)病率和死亡率呈逐年增長趨勢,這一趨勢在中國尤為明顯。在前列腺癌的治療中,早期患者通過前列腺切除術(shù)合并輔助放射來治療;對于晚期患者來說,主要為激素治療,但經(jīng)過一段治療期后,前列腺癌從對激素依賴逐漸變?yōu)榧に氐挚剐?這會使治療更加困難。最后當(dāng)去勢治療或傳統(tǒng)的激素治療失敗后,就只能采用化療。然而多次化療會使患者對化療藥物產(chǎn)生耐藥性,導(dǎo)致化療總體療效不佳。因此,開發(fā)更加有效的化療藥物是當(dāng)前治療前列腺癌的新趨勢。由于藥物發(fā)現(xiàn)和開發(fā)是一個昂貴和耗時的過程,以及舊藥新用給我們提供的捷徑,所以前期本實驗利用約翰·霍普金斯大學(xué)建立的臨床藥庫進(jìn)行大規(guī)模篩選,初步鑒定出一種目前已用于臨床治療呼吸道感染的抗生素藥物—氯�？朔�(Clofoctol),它能夠抑制前列腺癌激素不敏感細(xì)胞系PC3增殖。鑒于單藥治療時劑量增加造成的副反應(yīng)以及藥物聯(lián)用治療腫瘤的優(yōu)勢,前期本實驗室選用四十種在臨床上已經(jīng)應(yīng)用且對前列腺癌細(xì)胞有抑制作用的小分子藥物,與氯�？朔�(Clofoctol)分別聯(lián)用處理PC3細(xì)胞,根據(jù)Chou-Talalay計算公式及劑量-反應(yīng)實驗進(jìn)行篩選,表明氯�？朔�(Clofoctol)和索拉非尼(Sorafenib)能夠協(xié)同抑制PC3細(xì)胞的增殖。索拉非尼(Sorafenib)是一種多靶點多激酶抑制劑,經(jīng)美國FDA批準(zhǔn)被用于治療晚期腎細(xì)胞癌和肝癌,目前大量研究證實索拉非尼(Sorafenib)對前列腺癌的增殖具有抑制作用。本研究我們以前列腺癌PC3為細(xì)胞模型,研究氯�？朔�(Clofoctol)和索拉非尼(Sorafenib)協(xié)同作用的機(jī)制。通過細(xì)胞凋亡、細(xì)胞周期、Western blot以及qRT-PCR等技術(shù),我們確定10μM氯�？朔�(Clofoctol)和6μM索拉非尼(Sorafenib)聯(lián)合用藥能夠使78.84%的細(xì)胞被阻滯在G1期,且細(xì)胞協(xié)同凋亡率達(dá)到16.65%,高于Clofoctol或Sorafenib單獨用藥時的凋亡率;qRT-PCR結(jié)果顯示藥物聯(lián)用后ER-stress通路中DDIT3/CHOP、GADD34、ATF6和ATF4 mRNA表達(dá)水平明顯增高,對應(yīng)的Western blot結(jié)果表明8小時處DDIT3/CHOP和ATF4的蛋白水平顯著性協(xié)同上調(diào),并且在聯(lián)合處理24h小時處,XBP1前體明顯被剪接成有轉(zhuǎn)錄活性的XBP1s。也就是說,Clofoctol和Sorafenib聯(lián)用激活了ER-stress的IRE-1a和ATF6通路。接下來我們又觀察到氯�？朔�(Clofoctol)單獨用藥后PC3細(xì)胞會出現(xiàn)類似RNAi技術(shù)沉默VCP/p97后的內(nèi)質(zhì)網(wǎng)液泡化和泛素化蛋白累積現(xiàn)象。鑒于VCP/p97在內(nèi)質(zhì)網(wǎng)相關(guān)的蛋白酶體系統(tǒng)中起重要作用,因此我們推測氯�？朔�(Clofoctol)可能靶向VCP/p97,抑制其功能的發(fā)揮�；趯嶒炇业牧硗庖粋�研究方向,我們還進(jìn)行了microRNA的單核苷酸多態(tài)性與肺癌易感性的關(guān)聯(lián)分析。肺癌是發(fā)病率和死亡率增長最快,對人群健康和生命威脅最大的惡性腫瘤之一,而遺傳因素是影響肺癌發(fā)病的重要原因之一。越來越多的研究證明,microRNA發(fā)揮癌基因或抑癌基因的功能,且microRNA的單核苷酸多態(tài)性與腫瘤易感性存在一定關(guān)聯(lián)。我們通過前期的統(tǒng)計分析及文獻(xiàn)查閱后以miR-4293rs12220909多態(tài)性位點為研究對象,擬通過病例-對照的研究方法來分析該mirSNP與中國人群肺癌易感性的關(guān)聯(lián)。結(jié)果表明,在我們所研究的998例肺癌病例和1471例正常對照組中,miR-4293rs12220909多態(tài)性位點與肺癌的發(fā)病風(fēng)險具有顯著相關(guān)性:突變基因型GC/CC能夠顯著降低肺癌的發(fā)病風(fēng)險(OR=0.687;95%CI=0.564-0.837),等位基因C是肺癌易感性的保護(hù)因素(OR=0.734;95%CI=0.616-0.874);對遺傳模型進(jìn)行分層分析后發(fā)現(xiàn),GC基因型在≥62歲、62歲、男性、吸煙、非吸煙、肺腺癌及肺鱗癌人群中均能降低肺癌患病風(fēng)險。接下來,我們對于miR-4293 rs12220909多態(tài)性位點降低肺癌的發(fā)病風(fēng)險進(jìn)行了機(jī)制研究,發(fā)現(xiàn)野生型miR-4293可能通過調(diào)控抑癌基因PRKAA1和ADAMTSL3來增強(qiáng)肺癌易感性,從而在肺癌的發(fā)生、發(fā)展中起到促進(jìn)作用。
[Abstract]:Prostate cancer is one of the common diseases in male reproductive system tumors. The incidence and mortality of prostate cancer are increasing year by year. This trend is particularly obvious in China. In the treatment of prostate cancer in patients with early by prostate resection combined with radiotherapy for advanced treatment; patients, such as hormone therapy, but after a period after the treatment period, from prostate cancer for hormone dependent became steroid resistant, which makes the treatment more difficult. Finally, chemotherapy is used only after the treatment of castration or the failure of traditional hormone therapy. However, multiple chemotherapy can cause drug resistance to chemotherapeutic drugs, resulting in the overall effectiveness of chemotherapy. Therefore, the development of more effective chemotherapeutic drugs is a new trend in the treatment of prostate cancer. Due to drug discovery and development is a costly and time-consuming process, and to provide us with new old drugs, so this experiment using the pre clinical pharmacy store established by Johns Hopkins University for large-scale screening, preliminary identification of an antibiotic has already been used in the clinical treatment of respiratory tract infection clofoctol (Clofoctol), it can inhibiting hormone insensitive prostate cancer cell line PC3 proliferation. In view of the single drug treatment dose increased adverse reactions caused by drug use and treatment of tumor early advantage, the laboratory with forty kinds of small molecule drugs in clinical application and has inhibitory effects on prostate cancer cells, and clofoctol (Clofoctol) respectively connected to use PC3 cells, according to the Chou-Talalay calculation formula and experiment dose response were selected that clofoctol (Clofoctol) and Sola Fini (Sorafenib) could inhibit the proliferation of PC3 cells. Sola Fini (Sorafenib) is a multi target multi kinase inhibitor, approved by the US FDA, which is used for the treatment of advanced renal cell carcinoma and liver cancer. Currently, a large number of studies have proved that Sola Fini (Sorafenib) has an inhibitory effect on the proliferation of prostate cancer. In this study, we studied the synergistic mechanism of chloramphenol (Clofoctol) and Sola Fini (Sorafenib) in the PC3 cell model of prostate cancer. Through cell apoptosis, cell cycle, Western blot and qRT-PCR technology, we identified 10 M clofoctol (Clofoctol) and 6 M (Sorafenib) combined with sorafenib treatment can make 78.84% of the cells were arrested in G1 phase, and the cell apoptosis rate of cooperation reached 16.65%, apoptosis was higher than that of Clofoctol or Sorafenib alone. Rate; qRT-PCR results showed that the drug combination of ER-stress after DDIT3/CHOP, GADD34, ATF6 pathway and ATF4 mRNA expression levels were significantly increased, the corresponding Western blot results showed that the protein level of 8 hours at DDIT3/CHOP and ATF4 were co upregulated, and in the combined treatment of 24h hours, XBP1 precursor was cut into a transcriptional activity XBP1s. That is to say, Clofoctol and Sorafenib are used to activate the IRE-1a and ATF6 paths of ER-stress. Next, we observed that after PC3 alone, RNAi cells appeared vacuolization and ubiquitin accumulation in endoplasmic reticulum after RNAi VCP/p97 silencing. In view of the fact that VCP/p97 plays an important role in endoplasmic reticulum related proteasome system, we speculate that chlorfokol (Clofoctol) may target VCP/p97 and inhibit its function. Based on another laboratory research direction, we also conducted an analysis of the association of single nucleotide polymorphisms in microRNA with susceptibility to lung cancer. Lung cancer is one of the most malignant tumors that cause the fastest growth of morbidity and mortality and the most serious threat to people's health and life. Genetic factors are one of the important factors that affect the incidence of lung cancer. More and more studies have demonstrated that microRNA plays the function of oncogene or tumor suppressor gene, and microRNA single nucleotide polymorphism is associated with tumor susceptibility. We analyzed the association between mirSNP and susceptibility to lung cancer in China by case control study. We used the miR-4293rs12220909 polymorphic loci as the object of study. The results showed that in 998 cases of lung cancer that we study the cases and 1471 cases of normal control group, the risk of miR-4293rs12220909 polymorphism and lung cancer was significantly correlated with the mutation genotype GC/CC can significantly reduce the risk of lung cancer (OR=0.687; 95%CI= 0.564-0.837), C allele is a protective factor for lung cancer susceptibility (OR=0.734; 95%CI=0.616-0.874); hierarchical analysis of genetic model and found that the GC genotype can reduce the risk of lung cancer in more than 62 years old, 62 years old, male, smoking and non smoking, lung adenocarcinoma and squamous cell carcinoma of the lung in the crowd. Next, we carried out research on the mechanism of the miR-4293 rs12220909 polymorphism to reduce the risk of lung cancer, found that wild type miR-4293 can regulate the tumor suppressor gene PRKAA1 and ADAMTSL3 to increase the susceptibility to lung cancer, resulting in lung cancer occurrence and development play a role in promoting.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R737.25

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