本文選題：熊果酸　切入點：前列腺癌DU145細(xì)胞　出處：《青島大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：前列腺癌是老年男性常見惡性腫瘤,目前已成為第二位危害老年男性健康的惡性腫瘤。由于早期前列腺癌患者一般都無明顯臨床癥狀,大部分患者因臨床癥狀就診時,往往已發(fā)生遠(yuǎn)處骨轉(zhuǎn)移,失去根治性手術(shù)時機(jī)。大部分前列腺癌患者對初期雄激素剝奪的內(nèi)分泌治療效果明顯,但隨著病情進(jìn)展,患者仍會不可避免的發(fā)展為去勢抵抗性前列腺癌階段,嚴(yán)重影響患者生活質(zhì)量及生存時間,最終導(dǎo)致死亡。目前對于去勢抵抗性前列腺癌,缺乏標(biāo)準(zhǔn)有效的治療手段,因此迫切需要尋找有效的治療方法及藥物來延緩疾病進(jìn)展。中國的傳統(tǒng)中醫(yī)藥歷史悠久,應(yīng)用廣泛,在腫瘤治療領(lǐng)域也越來越引起大家的重視,具有藥效溫和、不良反應(yīng)少、毒副作用低等特點,已成為許多研究者關(guān)注的焦點。熊果酸是一種五環(huán)三萜烯類化合物,廣泛存在于杜鵑花科植物的葉子和果實以及毛地黃科、泡桐和木犀科女貞的葉子中。熊果酸具有廣泛的生物學(xué)活性,包括抗腫瘤、護(hù)肝、抗炎癥和抗病毒等。近年來的研究證明,熊果酸對多種惡性腫瘤細(xì)胞具有抑制增殖和誘導(dǎo)凋亡的作用,有研究報道,熊果酸對前列腺癌亦有較好的臨床治療效果。本實驗通過體外培養(yǎng)前列腺癌DU145細(xì)胞,研究和探討熊果酸對去勢抵抗性前列腺癌DU145細(xì)胞增殖的影響和促凋亡作用以及相關(guān)作用機(jī)制。用不同濃度的熊果酸作用于DU145細(xì)胞不同時間后,應(yīng)用CCK-8法、顯微鏡觀察、流式細(xì)胞術(shù)(FCM)、分光光度法、Western blot等多種方法觀察熊果酸對DU145細(xì)胞的影響,并探討相關(guān)作用機(jī)制,為治療前列腺癌尋找新的治療藥物提供實驗依據(jù)。目的通過體外培養(yǎng)去勢抵抗性前列腺癌DU145細(xì)胞,研究和探討熊果酸對DU145細(xì)胞增殖的影響和促凋亡作用以及相關(guān)作用機(jī)制。方法體外培養(yǎng)前列腺癌DU145細(xì)胞,用不同濃度的熊果酸(10、20、40、80μM)作用于DU145細(xì)胞不同時間進(jìn)行干預(yù),與對照組不加熊果酸相比,CCK-8法檢測熊果酸對DU145細(xì)胞增殖的抑制率;顯微鏡下觀察DU145細(xì)胞形態(tài)的改變;流式細(xì)胞術(shù)(FCM)檢測熊果酸對細(xì)胞凋亡率和細(xì)胞周期的影響;分光光度法檢測DU145細(xì)胞Caspase-3、Caspase-9活性的變化;Western blot分別檢測全細(xì)胞內(nèi)及細(xì)胞質(zhì)和線粒體中細(xì)胞色素C、cofilin-1的蛋白表達(dá),細(xì)胞質(zhì)中ROCK、PTEN的蛋白表達(dá)。用SPSS 13.0統(tǒng)計軟件對所得數(shù)據(jù)進(jìn)行統(tǒng)計學(xué)分析,計量資料以均數(shù)±標(biāo)準(zhǔn)差((?))表示,采用單因素方差分析進(jìn)行t檢驗,P0.05認(rèn)為差異具有統(tǒng)計學(xué)意義。結(jié)果1、CCK-8法測定熊果酸對DU145細(xì)胞的抑制作用,不同濃度的熊果酸(10、20、40、80μM)作用不同時間(24-72h)后,均能抑制DU145細(xì)胞的增殖,隨著熊果酸作用濃度的增加和作用時間的延長,其對DU145細(xì)胞增殖的抑制作用逐漸增強(qiáng),其中以80μM熊果酸作用72h后抑制效果最為明顯,呈明顯的濃度和時間依賴性。2、顯微鏡下觀察,熊果酸組細(xì)胞增殖緩慢,隨著熊果酸濃度的增加及作用時間的延長,出現(xiàn)體積變小、間隙增大、形態(tài)不規(guī)則、胞質(zhì)出現(xiàn)空泡、核仁濃縮、數(shù)目減少等細(xì)胞凋亡的形態(tài)學(xué)改變,而對照組細(xì)胞貼壁增殖良好,形態(tài)無明顯變化。3、流式細(xì)胞術(shù)檢測結(jié)果顯示,不同濃度熊果酸(10、20、40μM)作用DU145細(xì)胞48h后,細(xì)胞凋亡率分別為8.27±0.72%、12.84±0.75%、21.63±0.97%,呈明顯的濃度依賴性增高,與對照組凋亡率6.81±0.61%相比,20、40u M組有顯著統(tǒng)計學(xué)差異(P0.01)。細(xì)胞周期檢測結(jié)果顯示,累積于G0/G1期的細(xì)胞比例增多,而進(jìn)入S期的細(xì)胞比例減少,其中以40u M熊果酸組效果最為明顯,有顯著統(tǒng)計學(xué)差異(P0.01)。4、分光光度法對Caspase-3、Caspase-9活性檢測結(jié)果顯示,經(jīng)不同濃度的熊果酸(10、20、40μM)作用DU145細(xì)胞48h后,細(xì)胞中Caspase-3、Caspase-9活性逐漸增高,呈明顯濃度依賴性,有顯著統(tǒng)計學(xué)差異(P0.01)。5、Western blot分別檢測全細(xì)胞內(nèi)及細(xì)胞質(zhì)和線粒體中細(xì)胞色素C、cofilin-1蛋白表達(dá),結(jié)果顯示,用20μM和40μM熊果酸治療48h,DU145細(xì)胞的細(xì)胞質(zhì)中細(xì)胞色素C蛋白表達(dá)顯著增加、cofilin-1蛋白表達(dá)顯著降低(P0.01),而線粒體中細(xì)胞色素C蛋白表達(dá)顯著降低、cofilin-1蛋白表達(dá)顯著增高(P0.01);全細(xì)胞內(nèi)細(xì)胞色素C、cofilin-1蛋白表達(dá)基本不變。檢測胞質(zhì)中ROCK、PTEN蛋白表達(dá),結(jié)果顯示,UA可濃度依賴性誘導(dǎo)ROCK降解,ROCK蛋白表達(dá)梯度性降低,PTEN蛋白表達(dá)梯度性增高;對比對照組,20μM和40μM UA組細(xì)胞質(zhì)中ROCK蛋白表達(dá)顯著降低,PTEN蛋白表達(dá)顯著增高(P0.01),具有明顯濃度依賴性。結(jié)論1、熊果酸對DU145細(xì)胞增殖具有抑制作用,呈時間和濃度依賴性。2、熊果酸能濃度依賴性誘導(dǎo)DU145細(xì)胞凋亡,并參與細(xì)胞周期調(diào)控,使細(xì)胞阻滯于G0/G1期,S期數(shù)量降低。3、熊果酸可通過激活Caspase-9、Caspase-3級聯(lián)反應(yīng)的線粒體凋亡途徑誘導(dǎo)DU145細(xì)胞凋亡,其作用機(jī)制之一是UA可通過激活ROCK/PTEN信號通路介導(dǎo)的cofilin-1線粒體轉(zhuǎn)位而使線粒體釋放細(xì)胞色素C至細(xì)胞質(zhì)中,從而激活Caspase-9、Caspase-3級聯(lián)反應(yīng)的線粒體凋亡途徑,誘導(dǎo)DU145細(xì)胞凋亡。
[Abstract]:Prostate cancer is a common malignant tumor in elderly men, has become the second harm healthy elderly malignant tumor. Because patients with early prostate cancer generally have no obvious clinical symptoms, most patients with clinical symptoms, often have occurred distant bone metastasis, lost radical surgery time. Most patients with prostate cancer significantly to endocrine therapy the effect of early androgen deprivation, but as the disease progresses, the development will inevitably for patients with castration resistant prostate cancer stage, seriously affect the quality of life and survival time of the patients, eventually lead to death. At present for castration resistant prostate cancer, the lack of effective treatment, so it is urgent to find effective treatment methods and drugs to slow disease Chinese. Progress of traditional Chinese medicine has a long history, widely used in the field of cancer therapy more and more cause Everyone's attention, with moderate efficacy, less adverse reaction, low toxic and side effects, has become the focus of attention of many researchers. The ursolic acid is a pentacyclic three terpene compounds, widely exists in the Ericaceae plant leaves and fruits, foxglove, Paulownia and Oleaceae Ligustrum leaves of ursolic acid. Has a wide range of biological activities, including antitumor, anti-inflammatory and hepatoprotective, antiviral. Recent studies have shown that ursolic acid can inhibit proliferation and induce apoptosis in malignant tumors, studies have reported that ursolic acid also has better clinical curative effect on prostate cancer. The experiments of in vitro cultured prostate DU145 cancer cells, study and explore the effects of ursolic acid on castration resistant prostate cancer DU145 cell proliferation and apoptosis and related mechanism. With different concentrations of ursolic acid in DU14 5 different cells, using CCK-8 method, microscope, flow cytometry (FCM), spectrophotometry, Western, blot and other methods to observe the effects of ursolic acid on DU145 cells, and to explore the mechanism, to provide experimental basis for new therapeutics for the treatment of prostate cancer. Objective to search for castration resistant prostate cancer DU145 cells cultured in vitro, and to investigate the effects of ursolic acid on DU145 cell proliferation and apoptosis and related mechanism. Cultured prostate cancer DU145 cells in vitro, with different concentrations of ursolic acid (10,20,40,80 M) in DU145 cells at different time for intervention and control group without ursolic acid, inhibited on DU145 cell proliferation detection rate of ursolic acid by CCK-8; microscope was used to observe the morphological changes of DU145 cells; flow cytometry (FCM) detection of ursolic acid on the apoptosis rate and cell cycle. Ring; DU145 cells were detected by Caspase-3 spectrophotometry, the change of Caspase-9 activity; Western blot cells were detected in whole cell pigment and cytoplasmic and mitochondrial C, the expression of cofilin-1 protein in the cytoplasm of ROCK, expression of PTEN protein. The data were statistically analyzed by SPSS 13 statistical software, the measurement data to mean + the standard deviation ((?)) said that the analysis of t test was performed using ANOVA, P0.05 considered statistically significant. Results 1, inhibition of CCK-8 method for determination of ursolic acid on DU145 cells, different concentrations of ursolic acid (10,20,40,80 M) for different time (24-72h), can inhibit the DU145 cells with the increase of the proliferation of ursolic acid concentration and treatment time, the inhibitory effect on the proliferation of DU145 cells gradually increased, with 80 M of ursolic acid after 72h inhibition effect was the most obvious, obvious concentration And time dependent.2, under the microscope, ursolic acid group of cell proliferation, with increasing concentration and effect of ursolic acid in time, there are smaller, the increase of the gap, irregular shape, cytoplasmic vacuoles, nucleolus concentration, morphological changes of cell apoptosis and reduce the number of adherent cells, and the control group the proliferation of good, no obvious changes in morphology of.3, flow cytometry results showed that different concentrations of ursolic acid (10,20,40 M) DU145 cell 48h, cell apoptosis rates were 8.27 + 0.72%, 12.84 + 0.75%, 21.63 + 0.97%, depended on the concentration increased, compared with the control group, the apoptosis rate of 6.81 20,40u + 0.61%, M group with significant difference (P0.01). Cell cycle test results showed that the proportion of cells accumulated in G0/G1 phase increased, while the proportion of cells entering S phase decreased, which 40u M ursolic acid group was most effective, statistically The difference of Caspase-3.4 (P0.01), spectrophotometry, Caspase-9 activity assay showed that the different concentration of ursolic acid (10,20,40 M) DU145 48h cells, Caspase-3 cells, Caspase-9 activity gradually increased, in a dose dependent manner, with significant statistical difference (P0.01).5, Western blot cells were detected in whole cell pigment and cytoplasmic and mitochondrial C, cofilin-1 protein expression, results showed that 20 M and 40 M of ursolic acid in treatment of 48h, the expression of cytochrome C in the cytoplasm of DU145 cells was significantly increased, cofilin-1 protein expression decreased obviously (P0.01), and the expression of cytochrome C in mitochondria protein decreased expression of cofilin-1 protein was significantly increased (P0.01); whole cell cytochrome C, cofilin-1 protein expression unchanged. Detection of ROCK in the cytoplasm, the expression of PTEN protein. The results showed that UA concentration dependently induced ROCK degradation, ROCK The expression gradient decreased, PTEN protein expression gradient increased; compared with the control group, 20 M and 40 M UA group significantly decreased the expression of ROCK protein in the cytoplasm, the expression of PTEN protein was significantly increased (P0.01), with obvious concentration dependence. Conclusion 1, ursolic acid can inhibit the proliferation of DU145 cells in a time. And concentration dependent.2, ursolic acid can concentration dependent induction of DU145 cell apoptosis, and participate in the regulation of cell cycle, the cell cycle arrest in G0/G1 phase S phase, reducing the number of.3, ursolic acid can activate Caspase-9, mitochondrial apoptosis cascade reaction of Caspase-3 induced DU145 cell apoptosis, the mechanism of action is UA can be activated by ROCK/PTEN signaling pathway mediated by cofilin-1 mitochondrial translocation and release of cytochrome C from mitochondria to cytosol, which activates Caspase-9, mitochondrial apoptosis cascade reaction of Caspase-3, induced DU145 cell apoptosis Dead.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R737.25

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