熊果酸对前列腺癌DU145细胞体外增殖、凋亡的影响及机制的研究

发布时间：2018-03-14 11:42

本文选题：熊果酸　切入点：前列腺癌DU145细胞　出处：《青岛大学》2017年博士论文　论文类型：学位论文

【摘要】：前列腺癌是老年男性常见恶性肿瘤,目前已成为第二位危害老年男性健康的恶性肿瘤。由于早期前列腺癌患者一般都无明显临床症状,大部分患者因临床症状就诊时,往往已发生远处骨转移,失去根治性手术时机。大部分前列腺癌患者对初期雄激素剥夺的内分泌治疗效果明显,但随着病情进展,患者仍会不可避免的发展为去势抵抗性前列腺癌阶段,严重影响患者生活质量及生存时间,最终导致死亡。目前对于去势抵抗性前列腺癌,缺乏标准有效的治疗手段,因此迫切需要寻找有效的治疗方法及药物来延缓疾病进展。中国的传统中医药历史悠久,应用广泛,在肿瘤治疗领域也越来越引起大家的重视,具有药效温和、不良反应少、毒副作用低等特点,已成为许多研究者关注的焦点。熊果酸是一种五环三萜烯类化合物,广泛存在于杜鹃花科植物的叶子和果实以及毛地黄科、泡桐和木犀科女贞的叶子中。熊果酸具有广泛的生物学活性,包括抗肿瘤、护肝、抗炎症和抗病毒等。近年来的研究证明,熊果酸对多种恶性肿瘤细胞具有抑制增殖和诱导凋亡的作用,有研究报道,熊果酸对前列腺癌亦有较好的临床治疗效果。本实验通过体外培养前列腺癌DU145细胞,研究和探讨熊果酸对去势抵抗性前列腺癌DU145细胞增殖的影响和促凋亡作用以及相关作用机制。用不同浓度的熊果酸作用于DU145细胞不同时间后,应用CCK-8法、显微镜观察、流式细胞术(FCM)、分光光度法、Western blot等多种方法观察熊果酸对DU145细胞的影响,并探讨相关作用机制,为治疗前列腺癌寻找新的治疗药物提供实验依据。目的通过体外培养去势抵抗性前列腺癌DU145细胞,研究和探讨熊果酸对DU145细胞增殖的影响和促凋亡作用以及相关作用机制。方法体外培养前列腺癌DU145细胞,用不同浓度的熊果酸(10、20、40、80μM)作用于DU145细胞不同时间进行干预,与对照组不加熊果酸相比,CCK-8法检测熊果酸对DU145细胞增殖的抑制率;显微镜下观察DU145细胞形态的改变;流式细胞术(FCM)检测熊果酸对细胞凋亡率和细胞周期的影响;分光光度法检测DU145细胞Caspase-3、Caspase-9活性的变化;Western blot分别检测全细胞内及细胞质和线粒体中细胞色素C、cofilin-1的蛋白表达,细胞质中ROCK、PTEN的蛋白表达。用SPSS 13.0统计软件对所得数据进行统计学分析,计量资料以均数±标准差((?))表示,采用单因素方差分析进行t检验,P0.05认为差异具有统计学意义。结果1、CCK-8法测定熊果酸对DU145细胞的抑制作用,不同浓度的熊果酸(10、20、40、80μM)作用不同时间(24-72h)后,均能抑制DU145细胞的增殖,随着熊果酸作用浓度的增加和作用时间的延长,其对DU145细胞增殖的抑制作用逐渐增强,其中以80μM熊果酸作用72h后抑制效果最为明显,呈明显的浓度和时间依赖性。2、显微镜下观察,熊果酸组细胞增殖缓慢,随着熊果酸浓度的增加及作用时间的延长,出现体积变小、间隙增大、形态不规则、胞质出现空泡、核仁浓缩、数目减少等细胞凋亡的形态学改变,而对照组细胞贴壁增殖良好,形态无明显变化。3、流式细胞术检测结果显示,不同浓度熊果酸(10、20、40μM)作用DU145细胞48h后,细胞凋亡率分别为8.27±0.72%、12.84±0.75%、21.63±0.97%,呈明显的浓度依赖性增高,与对照组凋亡率6.81±0.61%相比,20、40u M组有显著统计学差异(P0.01)。细胞周期检测结果显示,累积于G0/G1期的细胞比例增多,而进入S期的细胞比例减少,其中以40u M熊果酸组效果最为明显,有显著统计学差异(P0.01)。4、分光光度法对Caspase-3、Caspase-9活性检测结果显示,经不同浓度的熊果酸(10、20、40μM)作用DU145细胞48h后,细胞中Caspase-3、Caspase-9活性逐渐增高,呈明显浓度依赖性,有显著统计学差异(P0.01)。5、Western blot分别检测全细胞内及细胞质和线粒体中细胞色素C、cofilin-1蛋白表达,结果显示,用20μM和40μM熊果酸治疗48h,DU145细胞的细胞质中细胞色素C蛋白表达显著增加、cofilin-1蛋白表达显著降低(P0.01),而线粒体中细胞色素C蛋白表达显著降低、cofilin-1蛋白表达显著增高(P0.01);全细胞内细胞色素C、cofilin-1蛋白表达基本不变。检测胞质中ROCK、PTEN蛋白表达,结果显示,UA可浓度依赖性诱导ROCK降解,ROCK蛋白表达梯度性降低,PTEN蛋白表达梯度性增高;对比对照组,20μM和40μM UA组细胞质中ROCK蛋白表达显著降低,PTEN蛋白表达显著增高(P0.01),具有明显浓度依赖性。结论1、熊果酸对DU145细胞增殖具有抑制作用,呈时间和浓度依赖性。2、熊果酸能浓度依赖性诱导DU145细胞凋亡,并参与细胞周期调控,使细胞阻滞于G0/G1期,S期数量降低。3、熊果酸可通过激活Caspase-9、Caspase-3级联反应的线粒体凋亡途径诱导DU145细胞凋亡,其作用机制之一是UA可通过激活ROCK/PTEN信号通路介导的cofilin-1线粒体转位而使线粒体释放细胞色素C至细胞质中,从而激活Caspase-9、Caspase-3级联反应的线粒体凋亡途径,诱导DU145细胞凋亡。
[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.

【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.25

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