| Background and objectiveGallbladder cancer is a malignant tumor which occur in the gallbladder(including the cystic duct,the fundus of the gallbladder,the body of the gallbladder,and the neck of the gallbladder).Because of lacking of specific symptoms in the early stage,prone to lymphatic metastasis and distant organ metastasis,most patients have been diagnosed at the advanced stage,and lost the opportunity of surgery.In addition,the traditional treatment methods like radiotherapy and chemotherapy have not shown significant advantages in the treatment of advanced gallbladder cancer,so patients still have a extremely poor prognosis and its a serious threatell to human health.Therefore,it is very important to explore new treatment to improve the prognosis of patients with gallbladder carcinoma.Vasculogenesis has been proved to be a promising therapeutic target in many tumors through vascular endothelial growth f-actor(VEGF)signaling pathway inhibitors,which makes the role of anti-angiogenic targeted therapy in the treatment of malignant cancer more and more concerned.Apatinib is a new type of small molecule tyrosine kinase inhibitor targeting angiogenesis.It is highly selective in inhibiting VEGFR-2 and has powerful effect to inhibit tumor angiogenesis and antitumor activity.Studies have shown that Apatinib has a good anti-tumor effect in various tumor transplantation models and well tolerance.In addition,several clinical trials have shown that Apatinib has a strong inhibitory effect on many advanced solid tumors,such as gastric cancer,non-small cell lung cancer,breast cancer,etc.Therefore,it is of great significance and value to explore the anti-tumor effect of apatinib on gallbladder carcinoma.Based on the mentioned above,this study was designed to establish a subcutaneous transplanted tumor model of human gallbladder cancer(GBC-SD)in nude mice which was treated with Apatinib at high and low dose levels and normal saline as a placebo control group.The research was aimed to investigate the effect of Apatinib on gallbladder cancer in vivo,which may lay the foundation of clinical application of Apatinib.Materials and MethodsHuman gallbladder cancer cell line GBC-SD cell were cultivated.Logarithmic growth GBC-SD cell were injected into the subcutaneous tissue of BALB/c-nu female nude mice(SPF level,5 to 6 weeks old).A successful xenotransplantation model of GBC-SD was established after 2 weeks.24 nude mice with similar tumor volume were randomly divided into 3 groups.The placebo group(n=8)was injected with normal saline once a day through gavage,the high doses Apatinib was injected with Apatinib(200 mg/kg)once a day through gavage,the low doses Apatinib was injected with Apatinib(100 mg/kg)once a day.To examine inhibitory effects of the transplanted tumor,tumor volume was estimated once every three days,and the rate of relative tumor growth was calculated once every three days too.Treatments continue for 14 days.The nude mice were dissected at 24 hours after completion of the last treatment,The subcutaneous tumor tissues of nude mice were completely resected,and the tissue of gallbladder cancer were embedded in paraffin wax.The morphology of transplanted tumor cells from gallbladder cancer was observed under light microscope.Immunohistochemical CD31 antibody was used to label tumor microvessel density(MVD),Ki67 to label tumor cell proliferation and VEGFR-2 mean optical density to label vascular endothelial growth factor receptor 2(VEGFR-2)to detect the antitumor effect of Apatinib.Results:1.The volume growth of transplanted tumor in the two treatment groups was significantly less than that in the placebo group on the 9th day after treatment (p<0.05).The tumor volume in the low dose group and the high dose group was significantly lower than that in the control group on the 12th and 15th day after treatment(p<0.05),And there was significant difference between the high dose group and the low dose group(p<0.05).2.After 2 weeks of administration,the relative tumor growth rates of.the low-dose and high-dose Apatinib groups were 70.21%and 59.46%,respectively.3.After 14 days of treatment,the micro vessel count in the 100 mg/kg Apatinib and 200 mg/kg Apatinib groups were significantly lower than those in the control group(19.98±2.76 VS 25.92±2.88,P=0.001;12.99±2.75 VS 25.92±2.88,P<0.001).There was significant difference in micro vessel count between the two experimental groups(12.99±2.75 VS 19.98±2.76,P=0.003).4.After 14 days of treatment,the percentage of Ki67 positive cells in the 100 mg/kg Apatinib group and 200 mg/kg Apatinib group was significantly lower than that in the blank control group(37.42±4.99 VS 53.28± 8.72,P=0.001;26.199±3.63 VS 53.28±8.72,P<0.001).There was significant difference in the Ki67 positive cell ratio between the two experimental groups(26.19±3.63 VS 37.42±4.99,P=0.003).5.After 14 days of treatment,the positive rate of VEGFR-2 antibody expression in 100 mg/kg apatinib group and 200 mg/kg Apatinib group was significantly lower than that in the blank control group(21.577±5.01 VS 32.56±4.43,P=0.001;15.23±2.13 VS 32.56±4.43,P<0.001),and there was.significant difference in the proportion of VEGFR-2 positive cells between the two experimental groups(15.23±2.13 VS 21.57±5.01,P--0.025).Conclusions:1.Apatinib could inhibit the growth of human gallbladder carcinoma GBC-SD cell line in nude mice,and the anti-tumor effect of 200 mg/kg apatinib group in nude mouse human tumor xenograft model is more obvious than 100 mg/kg apatinib group.2.Apatinib could inhibit the expression of VEGFR-2 in gallbladder carcinoma and reduce the micro vessel density of tumor and the proliferation of cancer cells. |
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