西黄丸抑制胃癌细胞增殖及其血管生成拟态形成的机制探讨

发布时间：2019-06-13 09:08

【摘要】：背景:我国是胃癌高发国家,胃癌发病率和致死率均位居我国肿瘤发病和死亡率前列,血管生成拟态是近年来新发现的血管生成形式,与胃癌的不良预后密切相关,而乏氧微环境是促进肿瘤血管生成拟态形成的主要因素之一,且与瘀毒互结证关系密切。抗癌名方西黄丸具有活血解毒功效并对多种肿瘤具有抑制作用,我们推测,具有解毒活血功效的西黄丸抑制肿瘤的可能机制是通过调控乏氧微环境进而抑制肿瘤血管生成拟态来实现的。目的:明确西黄丸调控乏氧微环境进而抑制肿瘤血管生成拟态形成的相关机制,探索西黄丸抗肿瘤的内在靶标,丰富活血解毒法治疗肿瘤的内涵。方法:(1)实验分组:空白组、西黄丸组、HIF-1α抑制剂(2-ME)组、结合组;(2)体外采用人胃癌MGC-803细胞系,CCK-8法检测西黄丸对肿瘤细胞增殖的抑制作用,AV-PI法检测西黄丸对肿瘤细胞凋亡的影响,RNA酶法检测西黄丸对肿瘤细胞周期的影响,体内实验采用Balb/c裸鼠建立MGC-803移植瘤模型,观察和测量西黄丸对裸鼠移植瘤肿瘤体积和质量的影响;(3)使用低氧工作站建立乏氧细胞培养模型、通过三维培养建立体外肿瘤细胞拟态血管模型,体外实验通过显微镜下管道计数、体内实验通过PAS-CD31免疫化学-免疫组化双染色法计算各组管道形成数目。(4)体内实验采用 RT-qPCR、Western-blot,体外实验采用 RT-qPCR、Western-blot、IHC法检测血管生成拟态相关蛋白VE-Cadherin、EphA2、MMP-2蛋白及mRNA表达、p-EphA2蛋白表达、HIF-1α、Twist1蛋白及mRNA表达情况。(5)HTA2.0芯片筛选西黄丸抗肿瘤和调控乏氧通路相关基因,并进行RT-qPCR验证。结果:(1)西黄丸可以抑制MGC-803细胞增殖,并呈浓度和时间依赖性,IC50:24h为50.424μg/ml,48h为35.129μg/ml,72h为28.191μg/ml,并抑制裸鼠移植瘤肿瘤的生长。同时,西黄丸可以促进MGC-803细胞凋亡,并将其细胞周期阻滞在G0/G1期。(2)建立体外和体内MGC-803细胞拟态管道形成模型,西黄丸可以显著抑制体外乏氧条件下和体内MGC-803拟态管道的形成,与2-ME的抑制作用相近,结合组对拟态管道形成的抑制作用最强。(3)西黄丸、2-ME、结合组可以降低体内和体外VE-Cadherin、MMP-2的mRNA表达,可以降低体内和体外VE-Cadherin、MMP-2的蛋白表达及p-EphA2的表达,结合组可以更显著的降低如上蛋白的mRNA和蛋白的表达。结合组可以降低EphA2的mRNA的表达,但不能显著影响其蛋白的表达;西黄丸、2-ME、结合组对EphA2蛋白和mRNA的表达没有显著影响。西黄丸、2-ME、结合组可以降低体外HIF-1α的mRNA和蛋白表达,降低Twist1的mRNA表达;在体内降低HIF-1α和Twist1的mRNA及蛋白表达。(4)HTA2.0基因芯片筛选西黄丸可能的作用通路靶点,发现西黄丸可以显著下调628个基因表达,上调88个基因表达。显著下调9个lncRNA表达,上调11个lncRNA表达,并对54条信号通路具有显著影响。结合前述研究结果选取乏氧信号通路和细胞周期信号通路进行验证,发现西黄丸可以降低HIF-1α上有蛋白mTOR表达,促进翻译后修饰蛋白PHD2、VHL蛋白基因表达,降低细胞周期相关蛋白(SMC3、CDC25c、CDC6、MCM3)基因的表达水平。结论:西黄丸可以通过(1)抑制乏氧相关mTOR-HIF-1α-Twistl信号通路,(2)促进乏氧相关蛋白PHD2、VHL表达,(3)抑制血管生成拟态相关蛋白VE-Cadherin、MMP-2表达及EphA2蛋白磷酸化,(4)抑制SMC3、CDC25c、CDC6、MCM3等细胞周期相关基因表达从而抑制人胃癌MGC-803细胞增殖及血管生成拟态形成的。
[Abstract]:BACKGROUND: China is a high incidence of gastric cancer. The morbidity and mortality of gastric cancer are in the forefront of the incidence and mortality of the tumor in China. The formation of angiogenesis is the newly discovered form of angiogenesis in recent years. It is closely related to the poor prognosis of gastric cancer. The hypoxic micro-environment is one of the main factors to promote the formation of tumor angiogenesis, and has a close relationship with the syndrome of blood stasis and poison. The anti-cancer Nifang pill has the effects of promoting blood circulation and removing toxic materials and inhibiting various tumors, and we have speculated that the possible mechanism of the western yellow pill with the effects of removing toxic substance and promoting blood circulation can be realized by regulating the hypoxic microenvironment and further inhibiting the formation of the tumor blood vessel. Objective: To study the mechanism of XHuangwan to regulate the hypoxic microenvironment and to inhibit the formation of tumor angiogenesis, and to explore the internal target of the anti-tumor of Xihuang Wan and enrich the connotation of the blood-activating and detoxicating method in the treatment of the tumor. Methods: (1) Experimental group: blank group, Xihuang pill group, HIF-1 inhibitor (2-ME) group and combination group; (2) human gastric cancer MGC-803 cell line and CCK-8 method were used in vitro to detect the inhibition of the proliferation of the tumor cells, and the AV-PI method was used to detect the effect of Xhuang pill on the apoptosis of the tumor cells. The effect of sihuang pill on the cell cycle of the tumor was detected by RNA enzyme method. In vivo, the tumor model of MGC-803 was established by Balb/ c nude mice, and the effect of Xihuang pill on the tumor volume and quality of the transplanted tumor in nude mice was observed and measured. (3) The hypoxic cell culture model was established by using the hypoxia workstation. The model of the in vitro tumor cell model was established by three-dimensional culture, and the number of tubes in each group was calculated by using a PAS-CD31 immunochemical-immunohistochemical double-staining method in vitro. (4) RT-qPCR, Western-blot and IHC were used to detect the expression of VE-Cadherin, EphA2, MMP-2 and the expression of mRNA, p-EphA2 protein, HIF-1, Twin1 and mRNA in the blood vessels by RT-qPCR, Western-blot and IHC. And (5) screening the anti-tumor and regulating the related genes of the hypoxia pathway by using the HTA2.0 chip, and carrying out RT-qPCR verification. Results: (1) Xihuang Wan can inhibit the proliferation of MGC-803 cells, and show the concentration and time-dependence, IC50:24 h is 50.424. mu. g/ ml,48 h is 35.129.mu. g/ ml, 72h is 28.191. mu. g/ ml, and the growth of transplanted tumor in nude mice is inhibited. At the same time, it can promote the apoptosis of MGC-803 cells and block the cell cycle in G0/ G1 phase. (2) In vitro and in vivo MGC-803 cell-like tube formation model, Xihuang pill can significantly inhibit the formation of the in vitro hypoxia condition and the in-vivo MGC-803 pseudo-state pipeline, which is similar to that of 2-ME, and the combination group has the strongest inhibition effect on the formation of the quasi-state pipeline. (3) The expression of VE-Cadherin, MMP-2 and the expression of p-EphA2 in the in-vivo and in-vitro VE-Cadherin and MMP-2 can be reduced and the expression of p-EphA2 can be reduced in vivo and in vitro. The binding group could decrease the expression of EphA2 mRNA, but could not significantly affect the expression of its protein. The expression of HIF-1 and Twin1 mRNA and protein in the in vitro expression of HIF-1 and Twin1 were decreased in Xhuang Pills,2-ME and combination group. (4) The gene chip of HTA2.0 was used to screen the possible pathway target of XHuangwan, and it was found that the expression of 628 genes could be reduced significantly and the expression of 88 genes was up-regulated. Significant down-regulation of 9 lncRNA expression, up-regulation of 11 lncRNA expression, and a significant effect on 54 signal pathways. in combination with that result of the above research, the hypoxia signal path and the cell cycle signal path are selected for verification, and the expression of the protein mTOR on the HIF-1 protein can be reduced, the expression of the post-translational modification protein PHD2 and the VHL protein gene is promoted, and the cell cycle related protein (SMC3, CDC25C, CDC6, MCM3) gene expression level. Conclusion: Xihuang Wan can inhibit the expression of hypoxia-related mTOR-HIF-1-Twistl signal, (2) promote the expression of hypoxia-related protein PHD2 and VHL, (3) inhibit the expression of the related protein VE-Cadherin, MMP-2 and EphA2 protein phosphorylation in the blood vessel, and (4) inhibit the expression of SMC3, CDC25C and CDC6. The cell cycle-related gene expression, such as MCM3, can inhibit the proliferation of MGC-803 cells and the formation of angiogenesis in human gastric cancer.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285

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