阿帕替尼联合参一胶囊抑制VEGFR-2高表达胃腺癌细胞SGC-7901增殖机制研究

Inhibitory Effects of Apatinib Combined With Shen Yi Capsule on the Proliferation of Gastric Adenocarcinoma Cells SGC⁃7901 and its Possible Mechanism

目的探讨阿帕替尼联合参一胶囊对VEGFR-2高表达的胃癌细胞增殖的影响。方法选择不同人胃腺癌细胞SGC-7901、BGS-823、AGS、MKN-45、MGC-83以及人脐静脉内皮细胞HUVEC(空白对照组)进行培养。RT-PCR筛选出VEGFR-2 mRNA水平高表达的细胞;western blotting检测VEGFR-2高表达胃腺癌细胞中相关蛋白表达量;实验药物分为空白对照组、阿帕替尼组、参一胶囊组、阿帕替尼联合参一胶囊组,除空白对照组外,每组设立7个浓度梯度;MTT法检测不同浓度下阿帕替尼、参一胶囊、阿帕替尼联合参一胶囊组对细胞增殖抑制率;利用中效原理绘制Fa-CI曲线,评价两药联合作用机制;流式细胞术检测空白对照组、阿帕替尼组、参一胶囊组、阿帕替尼+参一胶囊组中细胞凋亡率;免疫组化法检测凋亡相关蛋白Bcl-2、Bax、及死亡受体Fas蛋白的分布量。结果RT-PCR、western blotting结果显示胃腺癌细胞SGC-7901在VEGFR-2 mRNA、蛋白水平上均为高表达,为后期实验所选用。MTT结果显示两药效抑制细胞增殖,浓度为阿帕替尼:16μg/mL、参一胶囊:64μg/mL;根据药物中效原理,该浓度下药物联合指数CI<1,两药联合作用为协同。流式细胞术结果示阿帕替尼+参一胶囊组vs.空白对照组、阿帕替尼组、参一胶囊组其细胞凋亡率明显增高(P<0.0001),有显著统计学差异。免疫组化法检测发现阿帕替尼联合参一胶囊组vs.对照组和单药组,其凋亡相关蛋白Bcl-2、Bax、及死亡受体Fas分布量显著增加。结论阿帕替尼联合参一胶囊具有协同增效增强细胞的作用,其机制可能通过联合用药组调控细胞中抑凋亡基因Bcl-2、促凋亡基因Bax及死亡受体Fas蛋白表达,并激活线粒体途径和死亡受体Fas途径诱导细胞凋亡。

Objective To evaluate the inhibitory effects of apatinib combined with Shen Yi Capsules of VEGFR⁃2 high expression of gastric adenocarcinoma cells and explore its mechanism of promoting apoptosis.Methods Select different human gastric adenocarcinoma cells SGC⁃7901、AGS、MKN⁃45、MGC⁃803 and human venous cells HUVEC(blank control group)for cultivator⁃PCR was used to screens the gastric adenocarcinoma cell with high VEGFR⁃2 expression on mRNA levels.The expression of VEGFR⁃2 high expression of gastric adenocarcinoma cells detection of related proteins was explored by western blotting.The MTT assay was used to detect the inhibiting effect of different concentrations of apatinib,Shen Yi Capsules and apatinib combined with Shen Yi Capsules on the proliferation of VEGFR⁃2 high expression of gastric adenocarcinoma cells,to determine the effective inhibitory concentration of medicines.To utilize the principle of intermediate to draw the Fa⁃CI curves,to evaluate the mechanism of combined action between the apatinib and Shen Yi Capsule.Annexin V⁃FITC/PI was performed to investigate the apoptosis of VEGFR⁃2 high expression gastric adenocarcinoma cells at the group of control,apatinib,Shen Yi Capsule and apatinib combined with Shen Yi Capsule.Immunohistochemical methods was investigate the distribution of apoptosis related proteins about Bcl⁃2,Bax,Fas.Results The RT⁃PCT and western blotting results that the gastric adenocarcinoma cells SGC⁃7901 was all highly expressed on the levels of VEGFR⁃2 mRNA and protein(P<0.0001).The MTT showed that the inhibitory cell proliferation concentration was that apatinib:16μg/mL,Shen Yi Capsule:64μg/mL,and according to the principle of intermediate effect,the combination index CI<1,means that the combination of apatinib and Shen Yi Capsule was synergistic.Annexin V⁃FITC/PI results showed that the apoptosis rate of apatinib combined Shen Yi Capsule vs.apatinib,Shen Yi Capsule was significantly increased(P<0.05).Immunohistochemical methods was found that the group of apatinib combined Shen Yi Capsule increased the distribution of apoptosis⁃related proteins Bcl⁃2,Bax,Fas.Conclusions Apatinib combined with Shen Yi Capsule have synergistic inhibitory effect on gastric adenocarcinoma cells SGC⁃7901,and the synergistic mechanism maybe related to up⁃regulating expression of apoptosis related proteins that Bcl⁃2,Bax,Fas,and to induction of gastric apoptosis adenocarcinoma cells by sensitize the programmed mitochondrial death.