百里醌通过STAT3通路抑制体内外胃癌细胞增殖的研究

发布时间：2018-03-02 08:13

本文关键词： 百里醌胃癌 STAT3 增殖凋亡　出处：《武汉大学》2017年博士论文　论文类型：学位论文

【摘要】：第一部分百里醌在三种胃癌细胞中通过STAT3信号通路调节肿瘤细胞增殖凋亡的机制目的：探讨百里醌在三种胃癌细胞中是否通过STAT3通路调节肿瘤细胞增殖凋亡的机制。方法：将HGC27、BGC823、SGC7901细胞体外培养并用百里醌于每12小时、24小时、36小时作用于细胞(浓度梯度为0、10、25、50、75、100、125μmol/L)),并以MTT实验法检测细胞增殖抑制率,Western blot方法检测,分析STAT3蛋白、p-STAT3蛋白、STAT5蛋白表达量。结果：百里醌抑制STAT3在所有这些细胞中活化,以最大抑制发生在TQ浓度为50μM时且并没有影响STAT3蛋白的表达。免疫印迹清晰表明TQ在HGC27细胞细胞核中阻碍了STAT3核易位。TQ没有抑制STAT5磷酸化或STAT3蛋白的表达。结果表明,TQ专门抑制STAT3酪氨酸磷酸化。结论：在这三种胃癌细胞中TQ均通过抑制STAT3络氨酸磷酸化调节细胞凋亡,STAT3蛋白表达没有改变第二部分在HGC27胃癌细胞中STAT3信号通路调节肿瘤细胞增殖凋亡的相关基因目的：探讨HGC27胃癌细胞中哪些基因在STAT3信号通路中起决定性作用方法：本部分以胃癌细胞HGC27为研究对象,经RT-PCR、STAT3-荧光素酶报告基因分析、MTT、Hoechst 33258、膜联蛋白V细胞凋亡染色分析检测。STAT3, p-STAT3, STAT5, p-STAT5, p-JAK2, JAK2, p-Src, Src, GAPDH, Lamin-A, survivin, Cyclin D, Bcl-2, Bax, PPAR, Caspase-3,7,9蛋白的表通过免疫印迹法Western blot检测。流式细胞术(FCM)检测细胞周期和细胞凋亡。在HGC27细胞株中,TQ诱导凋亡细胞死亡时存在剂量依赖相关性现象,并通过Annexin V-FITC/PI分析和Hoechst 33258法检测。与对照组相比,P0.05为差异有统计学意义。结果：TQ诱导下调STAT3的激活和减少JAK2和C-src的活动。TQ也下调STAT3相关基因的表达,如Bcl-2, cyclin D, surviving和VEGF,但激活caspase-3,7,9。 Annexin V-FITC/PI分析结果显示TQ以浓度依赖性模式诱导细胞凋亡。Hoechst33258染色结果可见包含核碎片的凋亡小体。结论：TQ在HGC27细胞中抑制STAT3相关基因的表达,包括细胞周期蛋白D1、 bcl-2、生存素、血管生成基因产物(VEGF)、Bcl-xL和存活素可能与PARP的分裂有关,TQ可浓度依赖性激活caspase-3,7,9。第三部分体内实验百里醌对胃癌细胞生物学行为影响的研究目的：本部分研究探讨百里醌在裸鼠体内对胃癌细胞皮下移植瘤作用的具体作用机制。方法：在体内,裸鼠建立胃癌皮下移植瘤模型,被分为四组,每笼四只。肿瘤模型建成后裸鼠随机分配为四组：(1)未经处理,等容生理盐水；(2)10毫克／公斤TQ；(3)20毫克/公斤TQ；(4)30毫克/公斤TQ。腹腔内注射给药,每周三次。每3天称重,卡尺测量大小。30天后处死、称重。TUNNEL法检测胃癌细胞数量。与对照组相比,P0.05为有统计学意义。结果：实验裸鼠处死后剥离肿瘤称重。四组重量为别为：(762.7±35.2mg) (647.8±33.9mg) (595.3±30.3mg) (305.1±34.8mg),P0.05,有统计学差异。结论：与对照组相比治疗后肿瘤明显降低重量和体积。
[Abstract]:Part one the mechanism of thyriquinone regulating tumor cell proliferation and apoptosis through STAT3 signaling pathway in three kinds of gastric cancer cells objective: to investigate whether thyriquinone regulates tumor cell proliferation and apoptosis through STAT3 pathway in three gastric cancer cells. Methods: HGC27BGC823 SGC7901 cells were cultured in vitro and treated with thyriquinone every 12 hours for 24 hours or 36 hours (concentration gradient was 0101055075 / 100 渭 mol 路L ~ (-1)). The cell proliferation inhibition rate was detected by MTT assay, and the inhibition rate of cell proliferation was determined by Western blot assay, and the proliferation inhibition rate of HGC27BGC823 / SGC7901 cell line was determined by Western blot. The expression of STAT3 p-STAT3 protein and STAT5 protein were analyzed. Results: thyriquinone inhibited the activation of STAT3 in all these cells. The maximum inhibition occurred when TQ concentration was 50 渭 M and did not affect the expression of STAT3 protein. Western blot showed that TQ blocked the translocation of STAT3 nucleus in HGC27 cells. TQ did not inhibit the expression of STAT5 phosphorylation or STAT3 protein. Conclusion: in these three gastric cancer cells, TQ regulates the expression of apoptotic STAT3 protein by inhibiting STAT3 complex phosphorylation. The second part of TQ does not change the STAT3 signal pathway in HGC27 gastric cancer cells. Objective: to investigate which genes play a decisive role in the STAT3 signaling pathway in HGC27 gastric cancer cells. The expression of MTT3, p-STAT3, STAT5, p-STAT5, p-JAK2, JAK2, p-Src, Src, GAPDH, Lamin-A, survivin, Cyclin D, Bcl-2, Bax, PPAR, Caspase-3PPAR, Caspase-79 protein were detected by Western flow cytometry (FCM). Cell cycle and apoptosis. TQ induced apoptosis in HGC27 cell line in a dose-dependent manner. Compared with the control group, Annexin V-FITC / Pi analysis and Hoechst 33258 assay showed that there was a significant difference. Results: 1 TQ induced down-regulation of STAT3 activation and decreased the activity of JAK2 and C-src. TQ also down-regulated the expression of STAT3 related genes. For example, Bcl-2, cyclin D, surviving and VEGF, but caspase-3 was activated. The results of Annexin V-FITC / Pi analysis showed that TQ induced cell apoptosis in a concentration-dependent manner. Hoechst33258 staining showed that apoptotic bodies containing nuclear fragments could be found in HGC27 cells. Conclusion: TQ inhibits the expression of STAT3 related genes in HGC27 cells. Including cyclin D1, bcl-2, survivin, Bcl-xL and survivin may be related to the division of PARP. TQ can activate caspase-3 in a concentration-dependent manner. Part III: study on the effect of thyriquinone on the biological behavior of gastric cancer cells in vivo: this part is to investigate the effect of thyriquinone on the biological behavior of gastric cancer cells. The specific mechanism of the effect of quinone on subcutaneous tumor transplantation of gastric cancer cells in nude mice. Methods: in vivo, Nude mice were divided into four groups, four in each cage. After the tumor model was established, nude mice were randomly assigned to four groups: 1) untreated. Isovolumetric normal saline 2mg / kg TQX 3N 20 mg / kg TQX 430mg / kg TQ.Intraperitoneal injection, three times a week, weighing every 3 days, the caliper measured the size of the drug and was executed after 30 days. The number of gastric cancer cells was measured by weighing. Tunel method. Compared with the control group, P05 was statistically significant. Results: the weight of the tumor was removed after the nude mice were killed. The weight of the four groups was 647.8 卤33.9mg) 595.3 卤30.3mg), there was statistical difference between the four groups and the control group. Conclusion: there is a statistical difference between the four groups. Tumor weight and volume were significantly reduced after treatment.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2

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