一种新的CD13小分子抑制剂抗肝癌的作用及其机制研究

发布时间：2018-02-28 10:23

本文关键词： 肝癌 CD13 Bestatin 4.1-3k　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的肝癌是全球发病率第五、死亡率第二的恶性肿瘤,严重威胁着人类的生命。当前,肝癌在全球的发病率呈上升趋势,中国新增肝癌病例和死亡人数均居世界首位,约占全球总数的50%左右。由于肝癌的预后不佳,因此,需要寻求新的治疗手段或药物。CD13,又称氨肽酶N(APN),是一种Zn2+依赖的位于细胞表面的金属蛋白酶,能够与从寡肽N末端切除氨基酸的糖蛋白结合。CD13在多种组织细胞均有表达,例如神经细胞、髓样细胞、内皮细胞以及成纤维细胞等。同时,CD13在急性髓性白血病和多种实体瘤组织中呈现高表达的现象。因此,该分子又被称作肿瘤标记分子。由于CD13分布范围广,且生物学功能多样,不仅参与多种生理病理过程,还可作为肝癌干细胞的标志分子。由于CD13分布广泛,在临床治疗中需要选择特异性强的靶向抑制剂进行治疗,这是研发新型CD13抑制剂所面临的最大挑战。由此,本研究利用分子生物学手段,通过体内外实验,对我院药物化学课题组合成的新的CD13小分子抑制剂4.1-3k在治疗肝癌中的作用进行了研究,并对其作用机制进行了探讨,为临床治疗肝癌以及研发靶向药物提供了参考。研究方法1.以肝癌细胞系为模型,采用流式细胞术检测CD13分子在不同细胞系表面的表达情况;2.采用 MTT 法检测 CD13 抑制剂对 HepG2、PLC/PRF/5 以及 HepG2.2.15细胞增殖能力的影响;PI单染、Annexin V/PI双染法分别检测CD13抑制剂对HepG2、PLC/PRF/5以及HepG2.2.15细胞周期和细胞凋亡的影响;荧光定量PCR及Western Blot实验检测细胞凋亡基因Bcl-2、Bax的表达变化;3.Transwell实验分析CD13抑制剂对HepG2、HepG2.2.15细胞迁移的影响;荧光定量PCR检测经CD13抑制剂处理的HepG2、HepG2.2.15细胞中基质金属蛋白酶MMP2、MMP9的表达;4.血管生成实验检测CD13抑制处理HepG2、PLC/PRF/5以及HepG2.2.15细胞后的血管生成情况;荧光定量PCR检测经CD13抑制剂处理的HepG2和HepG2.2.15细胞中与血管生成相关的VEGF、MCP-1等基因的表达;5.Western blot 实验检测 CD13 抑制剂对 HepG2、HepG2.2.15 细胞 MAPK、NF-κB以及STAT3信号通路基因表达;荧光定量PCR检测经CD13抑制剂处理的HepG2以及HepG2.2.15细胞中炎症因子IL-6、IL-10等基因的表达;6.流式细胞术检测DEN/CCL4诱导肝纤维化模型、HBV模型、肝癌细胞荷瘤模型小鼠各个器官中CD13的表达,统计经CD13抑制剂治疗后的荷瘤裸鼠的生存期;7.建立肝癌的肺转移模型,经CD13抑制剂治疗后观测肺组织的转移情况;研究结果1.CD13在不同肝癌细胞中表达情况不同,其中含S抗原或HBV基因组的肝癌细胞系中CD13表达较高。2.CD13抑制剂显著抑制肝癌细胞的增殖能力,对HepG2.2.15细胞的作用最为明显;在高表达CD13的肝癌细胞系中,4.1-3k的抑制效果强于对照药Bestatin。CD13抑制剂处理后,肝癌细胞凋亡比例增加,4.1-3k在高表达CD13的肝癌细胞系中促凋亡效果强于Bestatin,但对细胞周期无影响。3.CD13抑制剂能够抑制HepG2、HepG2.2.15细胞迁移,且作用效果强于Bestatin。同时,基质金属蛋白酶MMP2、MMP9在经CD13抑制剂处理后的细胞中表达下调。4.CD13抑制剂处理肝癌细胞后,新生血管的生成明显受到抑制。在CD13高表达的肝癌细胞系中,4.1-3k抑制血管生成作用明显优于Bestatin。同时,血管生成相关基因VEGF、MCP-1在CD13抑制剂处理后显著下调。5.经CD13抑制剂处理后,HepG2、HepG2.2.15细胞中MAPK、NF-κB以及STAT3信号通路相关基因的活化受到抑制;同时,细胞中炎症因子IL-6、IL-8表达显著下调。6.DEN/CCL4诱导的肝脏纤维化模型小鼠及HBV模型小鼠中,CD13在免疫负调细胞中表达较高。经CD13抑制剂治疗后,HCC荷瘤小鼠中肿瘤的生长受到抑制,生存期延长。7.在BALB/c肺转移模型小鼠中,经CD13抑制剂治疗后肺结节数目明显减少,且在4.1-3k组效果最为显著。结论及意义本文对新型的CD13小分子抑制剂4.1-3k的抗肝癌作用进行系统评价,发现4.1-3k可通过促进肿瘤细胞凋亡抑制肿瘤细胞生长,同时可以抑制肿瘤细胞的迁移和新生血管的形成,从而发挥有效的抗HCC作用。体内小鼠模型实验发现,4.1-3k能抑制肝癌生长和转移,大大提高荷瘤小鼠存活率。因此,CD13小分子抑制剂4.1-3k可作为治疗肝癌的候选药物,为研发更加高效治疗肝癌的新药物提供了实验数据和理论支持。
[Abstract]:The purpose of the study is the global liver cancer incidence rate of fifth, second of the mortality rate of malignant tumor, a serious threat to human life. At present, the incidence of HCC in the world is rising, Chinese new liver cancer cases and deaths were ranked first in the world, accounting for about 50% of the global total. Due to poor prognosis of hepatocellular carcinoma, therefore, need to seek treatment or drug.CD13, also known as aminopeptidase N (APN), is located on the cell surface of a metalloproteinase dependent Zn2+, can be combined with resection of glycoprotein amino acids from N terminal peptide.CD13 were expressed in various tissues, such as nerve cells, myeloid cells, endothelial cells and fibroblasts cell. At the same time, CD13 showed high expression in tissue of acute myeloid leukemia and some solid tumors. Therefore, the molecule is called tumor marker CD13. Because of wide distribution, and biological function Diversity, not only involved in a variety of physiological and pathological processes, also can be used as a molecular marker of liver cancer stem cells. Since CD13 is widely distributed in the clinical treatment to choose specificity of targeted inhibitors for treatment, this is the biggest challenge facing the development of novel CD13 inhibitors. Thus, this study by means of molecular biology, in vivo and in vitro the role of our hospital, pharmaceutical chemistry research group of the new 4.1-3k CD13 inhibitors in the treatment of hepatocellular carcinoma were studied, and the mechanism is discussed, for the clinical treatment of liver cancer and the development of targeted drugs provides a reference. 1. research methods on hepatoma cell line model, using flow cytometry cells were detected on the surface of the CD13 molecule expression in different cell lines; 2. were detected by MTT CD13 and the effect of PLC/PRF/5 inhibitor on HepG2, HepG2.2.15 cell proliferation; PI staining, Detection of CD13 inhibitor on HepG2 Annexin V/PI double staining, PLC/PRF/5 and HepG2.2.15 on cell cycle and apoptosis; fluorescence quantitative PCR and Western Blot assay, apoptosis gene Bcl-2, Bax expression changes; analysis of CD13 inhibitor on HepG2 3.Transwell experiment, migration of HepG2.2.15 cells; HepG2 fluorescence quantitative PCR detection with CD13 inhibitor the matrix metalloproteinase MMP2 in HepG2.2.15 cells, the expression of MMP9; 4. angiogenesis assay of CD13 inhibition treatment HepG2, PLC/PRF/5 and angiogenesis of HepG2.2.15 cells; fluorescence quantitative PCR detection with CD13 inhibitor HepG2 and HepG2.2.15 cells and the angiogenesis related gene expression of VEGF, MCP-1 and other CD13 inhibitors; detection 5.Western blot of HepG2 HepG2.2.15 MAPK cell experiment, and the expression of NF- kappa B and STAT3 signaling pathway gene; fluorescence quantitative PCR detection with CD13 inhibitor HepG2 and inflammatory factors in HepG2.2.15 cells IL-6, IL-10 gene expression; liver fibrosis model induced by DEN/CCL4 detection of 6. flow cytometry HBV model, expression of CD13 in hepatoma cells in tumor bearing mice in various organs, statistics by CD13 in nude mice after treatment with inhibitor survival; 7. models of hepatocellular carcinoma lung metastasis, metastasis by CD13 inhibitor after treatment observation of lung tissue; the results of 1.CD13 expression in different liver cancer cells in different HCC cell lines, which containing S antigen or HBV genome in CD13 high expression of.2.CD13 inhibitor significantly inhibited the proliferation of hepatocellular carcinoma cells, the effects on HepG2.2.15 cells obviously; in hepatocellular carcinoma cell line CD13 high expression, 4.1-3k inhibition effect is stronger than the control after drug treatment of Bestatin.CD13 inhibitor, apoptosis of hepatoma cells increased in proportion to 4.1-3k High expression in hepatocellular carcinoma cell line CD13 in the pro apoptotic effect is stronger than Bestatin, but.3.CD13 has no effect on cell cycle inhibitors can inhibit HepG2 and HepG2.2.15 cell migration, and the effect is stronger than that of Bestatin. at the same time, matrix metalloproteinase MMP2, MMP9 in the CD13 treated cells was down regulated in cells treated with.4.CD13 inhibitors. Angiogenesis was significantly inhibited in hepatocellular carcinoma cell lines with high expression of CD13, 4.1-3k inhibited the angiogenesis was significantly higher than that of Bestatin. at the same time, angiogenesis related gene VEGF, MCP-1 inhibitor treatment after CD13.5. was down regulated by CD13 inhibitor, HepG2, MAPK in HepG2.2.15 cells, activation of NF- kappa B and STAT3 signal pathway related genes was inhibited; at the same time, inflammatory factors in IL-6 cells, IL-8 expression was significantly reduced in liver fibrosis model in mice induced by.6.DEN/CCL4 and HBV. In mice, the expression of CD13 was higher in the negative immune cells. After treatment with a CD13 inhibitor, HCC tumor bearing mice inhibited the growth and prolong the survival of.7. in a mouse model of BALB/c lung metastasis, the inhibitor of CD13 after treatment significantly reduced the number of lung nodules, and in the 4.1-3k group had the best effect. Conclusion and the significance of this article on CD13 small molecule inhibitors of 4.1-3k novel anti-tumor effects of systematic evaluation found that 4.1-3k can promote the apoptosis of tumor cells by inhibiting the growth of tumor cells, and can inhibit the migration and angiogenesis of tumor cell formation, so as to play an effective role of anti HCC. Found that the mice experiment, 4.1-3k inhibited the growth of and metastasis of liver cancer, greatly improve the survival rate of tumor bearing mice. Therefore, small molecule inhibitors of CD13 4.1-3k can be used as a drug candidate for the treatment of liver cancer, for development of new drugs more effective in the treatment of hepatocellular carcinoma The material provides experimental data and theoretical support.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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