本文選題：前列腺癌 + 雷公藤紅素��； 參考：《哈爾濱工業(yè)大學(xué)》2017年碩士論文

【摘要】：前列腺癌的發(fā)病率在西方國(guó)家位于榜首、其死亡率位于男性因癌癥致死的第二位。雄激素剝奪是早期前列腺癌的首選治療手段,雖然最初反應(yīng)良好,但其中大部分會(huì)發(fā)展為去勢(shì)抵抗型前列腺癌,表現(xiàn)為雄激素非依賴性。大量臨床前研究顯示雷公藤紅素,傳統(tǒng)中藥雷公藤(Tripterygium wilfordii)的主要成分,對(duì)雄激素非依賴性前列腺癌的療效顯著。課題組前期研究結(jié)果表明,雷公藤紅素在雄激素受體(Androgen receptor,AR)陽(yáng)性的前列腺癌細(xì)胞中通過靶向AR誘導(dǎo)保護(hù)性細(xì)胞自噬。然而,自噬對(duì)細(xì)胞凋亡的影響因藥物的不同以及相同藥物與不同細(xì)胞相互作用的不同而發(fā)生改變。本課題以DU145與PC-3兩種雄激素非依賴的AR陰性前列腺癌細(xì)胞系為研究對(duì)象,探討雷公藤紅素誘導(dǎo)的細(xì)胞自噬與細(xì)胞凋亡的關(guān)系,為雷公藤紅素在雄激素非依賴性前列腺癌治療的臨床前研究提供實(shí)驗(yàn)依據(jù)。本課題通過Western Blotting實(shí)驗(yàn)明確了雷公藤紅素在AR陰性前列腺癌細(xì)胞中誘導(dǎo)細(xì)胞自噬與細(xì)胞凋亡。為探究雷公藤紅素誘導(dǎo)的細(xì)胞自噬與細(xì)胞凋亡的關(guān)系,分別采用細(xì)胞自噬與細(xì)胞凋亡抑制劑處理后檢測(cè)細(xì)胞凋亡與細(xì)胞自噬的水平變化。抑制細(xì)胞自噬后,DU145與PC-3對(duì)雷公藤紅素的敏感性增強(qiáng),凋亡水平上調(diào),表明雷公藤紅素在AR陰性前列腺癌細(xì)胞中誘導(dǎo)保護(hù)性細(xì)胞自噬。抑制細(xì)胞凋亡后,Western Blotting結(jié)果顯示在DU145與PC-3中,與雷公藤紅素單獨(dú)用藥相比,LC3-II/LC3-I、Atg7、Beclin1的表達(dá)水平均有不同程度的上調(diào),說明細(xì)胞自噬的啟動(dòng)過程被激活;p62蛋白在8-24 h均顯著上調(diào),說明抑制細(xì)胞凋亡阻滯自噬潮的進(jìn)行,表明細(xì)胞凋亡促進(jìn)細(xì)胞自噬。接下來探究在AR陰性前列腺癌細(xì)胞中,雷公藤紅素誘導(dǎo)細(xì)胞自噬的機(jī)制。首先,Western Blotting顯示雷公藤紅素誘導(dǎo)的自噬與Bcl-2/Beclin1相關(guān)通路無關(guān)。其次,通過q RT-PCR證實(shí)在DU145與PC-3中,雷公藤紅素抑制自噬抑制分子mi R-101的表達(dá)。HIF-1可結(jié)合在mi R-101轉(zhuǎn)錄起始位點(diǎn)上游,調(diào)控其表達(dá)。Western Blotting顯示雷公藤紅素處理引起HIF-1α蛋白上調(diào)。在DU145與PC-3中轉(zhuǎn)染HIF-1α質(zhì)粒,通過q RT-PCR證實(shí)HIF-1α抑制mi R-101的表達(dá)。綜上,本課題的實(shí)驗(yàn)結(jié)果表明在AR陰性的前列腺癌細(xì)胞中,雷公藤紅素促進(jìn)HIF-1α的表達(dá),造成mi R-101表達(dá)抑制,從而誘導(dǎo)細(xì)胞自噬,而抑制細(xì)胞自噬有利于增強(qiáng)雷公藤紅素在前列腺癌細(xì)胞中誘導(dǎo)的細(xì)胞凋亡。
[Abstract]:Prostate cancer is at the top of the list in the West and is the second leading cause of cancer deaths among men. Androgen deprivation is the preferred treatment for early prostate cancer. Although the initial response is good, most of them develop castrated resistant prostate cancer, showing androgen independent. A large number of preclinical studies showed that tripterygium wilfordii, the main component of tripterygium wilfordii, a traditional Chinese medicine, has a significant effect on androgen independent prostate cancer. The previous study results showed that tripterine induced autophagy by targeting AR in androgen receptor AR-positive prostate cancer cells. However, the effect of autophagy on apoptosis changes with different drugs and different interactions between the same drug and different cells. In this study, two androgen independent AR negative prostate cancer cell lines, DU145 and PC-3, were used to investigate the relationship between autophagy induced by tripterine and apoptosis. To provide experimental evidence for the preclinical study of Tripterygium wilfordii in the treatment of androgen-independent prostate cancer. In this study, Western Blotting assay showed that tripterine induced autophagy and apoptosis in AR negative prostate cancer cells. In order to investigate the relationship between autophagy and apoptosis induced by tripterine, the level of apoptosis and autophagy were detected by the treatment of autophagy and apoptosis inhibitor respectively. After inhibition of autophagy, the sensitivity of DU145 and PC-3 to tripterin was enhanced, and the level of apoptosis was up-regulated, which indicated that tripterin induced autophagy in AR negative prostate cancer cells. After inhibiting apoptosis, the results of Western Blotting showed that in DU145 and PC-3, compared with tripterine alone, the expression level of LL-3-IILC3-Itg7BL-Beclin1 was up-regulated in different degrees, which indicated that the activation of p62 protein in autophagy was significantly up-regulated in 8-24 h. The inhibition of apoptosis blocked the development of autophagy, suggesting that apoptosis promoted autophagy. The mechanism of autophagy induced by tripterine in AR-negative prostate cancer cells was then explored. Firstly, Western Blotting showed that the autophagy induced by tripterine was independent of Bcl-2/Beclin1 related pathway. Secondly, it was confirmed by Q RT-PCR that tripterine inhibited the expression of autophagy inhibitor mi R-101 in DU145 and PC-3. HIF-1 could bind to the upstream of the transcription initiation site of miR-101. Western Blotting showed that tripterine treatment induced the up-regulation of HIF-1 偽 protein. After transfection of HIF-1 偽 plasmid into DU145 and PC-3, Q RT-PCR confirmed that HIF-1 偽 inhibited the expression of miR-101. In conclusion, our results show that in AR negative prostate cancer cells, tripterine promotes the expression of HIF-1 偽 and induces autophagy by inhibiting the expression of miR-101. Inhibition of autophagy can enhance the apoptosis induced by tripterine in prostate cancer cells.
【學(xué)位授予單位】：哈爾濱工業(yè)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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