【作者】 王玲；

【导师】 杨扬；

【作者基本信息】 桂林医学院 ， 药学（专业学位）， 2019， 硕士

【摘要】 目的:本文通过研究口服小分子抗肿瘤血管生成药物阿帕替尼体内外对肝癌Hep3B细胞的抑制作用,并探讨其抑制肝癌细胞增殖的作用机制,为阿帕替尼作为肝癌化疗药物的临床研究提供理论和实验依据。方法:1.体外实验:（1）CCK8法筛选用于实验的肝癌细胞;（2）CCK8法检测阿帕替尼对肝癌Hep3B细胞活性的影响;（3）流式细胞仪检测阿帕替尼对肝癌Hep3B细胞凋亡的影响;（4）荧光显微镜下观察阿帕替尼对肝癌Hep3B细胞线粒体膜电位的影响;（5）western blot检测阿帕替尼对蛋白USP22、Parkin、MCL1在肝癌Hep3B细胞中表达的影响。2.体内实验:（1）阿帕替尼对Hep3B细胞移植瘤的抑瘤作用;（2）HE染色观察阿帕替尼对细胞形态学的影响;（3）免疫组化法观察阿帕替尼对蛋白USP22、Parkin、MCL1在肿瘤组织中表达的影响。体内外实验中均以索拉菲尼作为对照药物。结果:1.经阿帕替尼（30μmol/L）作用24 h后细胞活性最低的肝癌Hep3B细胞作为本实验的研究对象;2.阿帕替尼和索拉菲尼对肝癌Hep3B细胞有较好的抑制作用,且抑制作用与时间、浓度呈正相关。作用24 h,阿帕替尼IC₅₀>25μmol/L,索拉菲尼IC₅₀=13.17±1.23μmol/L;作用48 h,阿帕替尼IC₅₀=18.23±0.89μmol/L,索拉菲尼IC₅₀=5.11±0.98μmol/L;作用72 h,阿帕替尼IC₅₀=9.87±0.57μmol/L,索拉菲尼IC₅₀<5μmol/L。3.阿帕替尼和索拉菲尼均能促进肝癌Hep3B细胞凋亡,凋亡率随着药物浓度的增大而增大;阿帕替尼（0μmol/L、15μmol/L、20μmol/L、25μmol/L）凋亡率分别是5.73±0.8%、7.8±9.76%、16.9±2.62%、21.1±1.08%,索拉菲尼（0μmol/L、5μmol/L、10μmol/L、15μmol/L）凋亡率分别是5.9±0.46%、7.3±0.89%、14.5±1.13%、29.83±5.37%。4.阿帕替尼和索拉菲尼均能使细胞线粒体膜电位下降,药物浓度越大,线粒体膜电位下降越大。5.阿帕替尼和索拉菲尼均会影响蛋白USP22、Parkin、MCL1蛋白表达量,药物浓度越大,作用时间越长,蛋白USP22、Parkin、MCL1下调越明显。6.阿帕替尼和索拉菲尼均能抑制肝癌移植瘤的生长;阿帕替尼50 mg/kg抑瘤作用最明显,索拉菲尼30 mg/kg和阿帕替尼30 mg/kg作用较接近。7.给药组细胞数目较少,细胞间隙明显增大,胞间存在较多空泡,阿帕替尼50mg/kg形态学变化最明显。8.给药组蛋白USP22、Parkin、MCL1表达量均下调,阿帕替尼50 mg/kg下调最明显,阿帕替尼30 mg/kg和索拉菲尼30 mg/kg各蛋白表达量相当。结论:（1）阿帕替尼或索拉菲尼时间和剂量依赖的方式抑制人肝癌Hep3B细胞增殖;（2）阿帕替尼或索拉菲尼可通过USP22/Parkin/MCL1途径抑制肝癌细胞增殖。更多还原

【Abstract】 Objective:The inhibitory effect of Apatinib which is oral small molecule anti-tumor angiogenesis drug was investigated on Hep3B cell in vitro and in vivo,and its mechanism of inhibiting the proliferation of hepatoma cells was explored.It is provided a theory and experimental basis for the clinical study of Apatinib as a chemotherapy drug for liver cancer.Methods:1.In vitro:（1）The cell lines were screened for experiments by CCK8 method;（2）The viabilities of Apatinib on Hep3B cell were detected by CCK8 method;（3）The apoptosis rates of Apatinib on Hep3B cell were examinated by flow cytometry;（4）The change of mitochondrial membrane potential of Apatinib on Hep3B cell was observed by JC-1 staining;（5）The expression levels of protein USP22,Parkin and MCL1 were tested on Hep3B cell treated with Apatinib by western blot.2.In vivo:（1）The animal model of human Hepatocellular carcinoma Hep3B cell transplanted in nude was estabilished to test anticancer effect of Apatinib;（2）HE staining was used to observe the effect of tumor and normal liver tissue cell morphology in untreated and Apatinib-treated group;（3）The expression levels of the protein USP22,Parkin and MCL1 were detected by immunohistochemistry experiment in untreated and Apatinib-treated group.Sorafenib was used as a contrast drug in the experiment.Results:1.Hep3B cell was the lowest viability cell treated with Apatinib（30μmol/L）for 24 h.It was used as the research object of this experiment.2.Apatinib had good inhibitory effect on Hep3B cell.The inhibition rates were positively correlated with time and dose.It discovered that IC₅₀ values of Hep3B cell were exceeded 25μmol/L,18.23±0.89μmol/L,9.87±0.57μmol/L for 24 h,48 h,72 h treated with Apatinib,respectively.And the IC₅₀ values of Hep3B cell were 13.17±1.23μmol/L,5.11±0.98μmol/L,less than 5μmol/L for 24 h,48 h,72 h treated with Sorafenib,respectively.3.Apatinib or Sorafenib could promote the Hep3B cell apoptosis.The apoptosis rates were increased in a dose-dependent manner.The apoptotics rates of Apatinib（0μmol/L,15μmol/L,20μmol/L,25μmol/L）were 5.73±0.8%,7.83±0.76%,16.9±2.62%,21.1±1.08%,and the apoptosis rates of Sorafenib（0μmol/L,5μmol/L,10μmol/L,15μmol/L）were 5.9±0.46%,7.3±0.89%,14.5±1.14%,29.83±5.37%,respectively.4.Apatinib or Sorafenib could decrease the mitochondrial membrane potential of Hep3B cell.The mitochondrial membrane potential also decreased in a dose-dependent manner.5.Apatinib or Sorafenib could down-regulated the expression levels of protein USP22,Parkin and MCL1 protein in a time-and dose-dependent manner on Hep3B cell.6.Apatinib or Sorafenib could inhibit the growth of liver cancer transplanted tumors.The anti-tumor effect of Apatinib（50 mg/kg）was the most obvious,which of Sorafenib and Apatinib were closer in the dose of 30 mg/kg.7.The numbers of cells were decreased,and the cell gaps were significantly increased.There were more vacuoles between the cells in the Apatinib or Sorafenib-treated group.The morphological changes of Apatinib（50 mg/kg）were the most obvious.8.The expression levels of protein USP22,Parkin and MCL1 were down-regulated in the Apatinib or Sorafenib-treated group,which of Apatinib（50mg/kg）was the most obvious.The protein USP22,Parkin and MCL1 expression levels of Apatinib or Sorafenib in the dose of30mg/kg were equivalent.Conclusions:（1）Apatinib or Sorafenib inhibited the proliferation of human liver cancer cells and tumors in a time-and dose-dependent manner;（2）Apatinib or Sorafenib inhibited liver cancer cells proliferation may through the USP22/Parkin/MCL1single pathway.更多还原