穿心莲内酯对胆囊癌的抑制作用及其分子机制的研究

发布时间：2018-08-25 10:38

【摘要】：目的:该课题探索穿心莲内酯对胆囊癌GBC-SD及NOZ细胞在体内、外的抑制作用及其相关机制,从而为穿心莲内酯成为一种具有潜在应用价值的新型胆囊癌治疗药物提供理论依据。方法:通过MTT实验、细胞单克隆实验探究穿心莲内酯对胆囊癌细胞增殖的抑制作用;利用流式细胞仪检测和Hoechst 33342染色分析穿心莲内酯对胆囊癌细胞凋亡及细胞周期的影响;利用细胞划痕实验及Transwell小室实验探究穿心莲内酯对胆囊癌细胞转移性的影响作用;通过裸鼠皮下成瘤实验检测穿心莲内酯在体内对胆囊癌细胞成瘤的抑制作用。通过Western blot实验检测在穿心莲内酯作用后凋亡及周期相关蛋白表达情况,并通过qRT-PCR实验、Western blot实验及免疫组化染色探讨穿心莲内酯作用于胆囊癌细胞后体内及体外的相关分子机制。结果:结果表明穿心莲内酯有抑制胆囊癌细胞增殖作用,且呈现出剂量和时间依赖性。穿心莲内酯可以诱导胆囊癌细胞的凋亡,并出现明显的核固缩、凋亡小体形成的形态改变。在蛋白水平上,Bcl-2水平下降,Bax水平升高,促进线粒体内细胞色素C向胞质释放,激活下游的caspase-9,caspase-3等,诱发线粒体介导的凋亡途径。穿心莲内酯治疗后胆囊癌细胞中处于S期的细胞比例升高,诱发S期细胞周期停滞,cyclin A及cyclin D1表达下降,裸鼠成瘤实验表明,穿心莲内酯治疗组裸鼠成瘤体积及重量比对照组都明显减小,对于穿心莲内酯对胆囊癌作用机制的研究我们发现,穿心莲内酯可以在体内和体外抑制胆囊癌细胞JAK2-STAT3信号通路。结论:我们的研究结果揭示穿心莲内酯在胆囊癌细胞中的抗癌作用,并证实穿心莲内酯激活胆囊癌GBC-SD和NOZ细胞线粒体介导的凋亡途径、诱导S期周期停滞和抑制胆囊癌细胞的转移特性。此外,我们的研究表明该药物可以抑制NOZ细胞裸鼠皮下种植瘤的生长。对穿心莲内酯在胆囊癌中的抗癌机制研究,我们发现其对胆囊癌的抗癌作用可能与抑制JAK2/STAT3信号通路有关。这些结果为穿心莲内酯成为一种具有潜在应用价值的胆囊癌治疗药物提供基础理论依据。
[Abstract]:Objective: to explore the inhibitory effect of andrographolide on GBC-SD and NOZ cells in vivo and in vitro and its related mechanism, so as to provide a theoretical basis for andrographolide to become a new type of therapy for gallbladder carcinoma with potential application value. Methods: the inhibitory effect of andrographolide on the proliferation of gallbladder cancer cells was investigated by MTT assay and monoclonal assay, and the effects of andrographolide on apoptosis and cell cycle of gallbladder cancer cells were analyzed by flow cytometry and Hoechst 33342 staining. The effect of andrographolide on the metastasis of gallbladder cancer cells was investigated by cell scratch test and Transwell chamber test. The inhibitory effect of andrographolide on tumor formation of gallbladder cancer cells in vivo was detected by subcutaneous tumorigenesis test in nude mice. Apoptosis and expression of cycle related proteins were detected by Western blot assay. The molecular mechanism of andrographolide acting on gallbladder cancer cells in vivo and in vitro was investigated by qRT-PCR blot assay and immunohistochemical staining. Results: the results showed that andrographolide inhibited the proliferation of gallbladder cancer cells in a dose and time dependent manner. Andrographolide could induce apoptosis of gallbladder cancer cells, with obvious nuclear pyknosis and morphological changes of apoptotic bodies. At the protein level, the level of Bcl-2 decreased and Bax increased, which promoted the release of cytochrome C into cytoplasm, activated downstream caspase-9,caspase-3, and induced mitochondria mediated apoptosis. After treatment with andrographolide, the proportion of cells in S phase increased and the expression of cyclin A and cyclin D1 decreased in S phase cell cycle arrest. The tumorigenic volume and weight of nude mice in andrographolide treatment group were significantly lower than those in control group. We found that andrographolide could inhibit the JAK2-STAT3 signaling pathway of gallbladder cancer cells in vivo and in vitro after the study of the mechanism of andrographolide on gallbladder carcinoma. Conclusion: our results reveal the anticancer effect of andrographolide in gallbladder cancer cells and demonstrate that andrographolide activates mitochondrial apoptosis in GBC-SD and NOZ cells. To induce phase S cycle arrest and inhibit metastasis of gallbladder cancer cells. In addition, our study suggests that the drug can inhibit the growth of subcutaneous implanted tumor in NOZ cells in nude mice. To study the anticancer mechanism of andrographolide in gallbladder carcinoma, we found that the anticancer effect of andrographolide on gallbladder carcinoma may be related to the inhibition of JAK2/STAT3 signaling pathway. These results provide a theoretical basis for andrographolide as a potential therapeutic drug for gallbladder carcinoma.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.8

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