一个新的IL6R抗体“HC6R1”抗肝癌的研究

发布时间：2018-05-16 14:00

本文选题：肝细胞肝癌 + HC6R1　；参考：《苏州大学》2016年硕士论文

【摘要】：目的:1.鉴定HC6R1的特异性及其识别位点;2.研究HC6R1对肝细胞肝癌的抗瘤作用及其机制。方法:用人肝癌组织免疫小鼠,通过杂交瘤技术制备抗体。采用流式细胞术和免疫组化检测HC6R1是否能够特异性识别肝癌细胞和组织。通过免疫共沉淀及质谱分析鉴定HC6R1的识别位点。构建载体转染细胞使得IL6R基因沉默,以HC6R1和anti-IL6R作为一抗,通过Western blotting和流式细胞术进一步证明其识别位点。利用一系列的体外实验如CCK8、划痕实验、粘附实验和Transwell分别检测HC6R1对肝癌细胞的增殖、迁移、粘附和侵袭能力的影响;通过皮下注射HepG2和Huh7细胞建立肿瘤模型研究HC6R1对体内肿瘤的抑制作用。采用Annexin V/PI双染和流式细胞术检测HC6R1对肝癌细胞凋亡和细胞周期的影响。利用基因芯片技术检测HC6R1作用的靶基因。以STAT3、p-STAT3、CyclinD1和CDK4抗体作为一抗,用Western blotting检测其靶向基因的蛋白质表达水平。结果:1.HC6R1的制备和鉴定1.1利用杂交瘤技术制备并筛选出一种阳性单克隆抗体即HC6R1。1.2对人肝癌细胞系/肝癌组织和其他细胞/组织进行流式细胞术和免疫组化检测发现:HC6R1可以被人肝癌细胞和肝癌组织识别,而在正常肝脏细胞和癌旁组织中几乎检测不到HC6R1。1.3通过免疫共沉淀及质谱分析发现HC6R1抗体识别的抗原可能是IL6R。采用Western blotting和流式细胞术检测发现肝癌细胞内IL6R基因敲低后,HC6R1抗原和anti-IL6R的表达量均明显减少,进一步证实其识别的抗原为肝癌细胞的IL6R。2.HC6R1对肝癌的抗瘤作用及其机制研究2.1 CCK8检测发现HC6R1处理组细胞的增殖速度明显慢于mIgG处理的对照组;划痕实验结果显示HC6R1能够显著抑制肝癌细胞的迁移,粘附实验和Transwell实验发现HC6R1能够显著抑制肝癌细胞的粘附和侵袭能力。2.2.利用肝癌细胞对小鼠体内成瘤实验发现,HC6R1可以抑制小鼠皮下肿瘤的生长;同时可以抑制原位成瘤小鼠肿瘤的生长并有效延长原位成瘤小鼠的生存时间。2.3通过检测细胞凋亡和细胞周期发现,HC6R1可以促进肝癌细胞细胞的凋亡,并且能够将细胞周期阻滞在G1/S期,从而抑制肿瘤细胞的增殖。利用基因芯片技术检测HC6R1作用的靶基因发现Bcl-2,Bcl-x L,Survivin,Myc,Cyclin D1,VEFG,HIF-1,MMP-2,IL-6和IL-10基因显著活化,其中大部分基因是STAT3信号通路的下游基因。通过Western blotting检测其靶向基因的蛋白质表达水平发现HC6R1抗体可以下调HepG2和Huh7细胞p-STAT3、CyclinD1和CDK4的基因表达水平。结论:1.HC6R1能够特异性识别人肝癌细胞和组织,其作用位点为IL6R。2.HC6R1能够显著抑制肝癌细胞的增殖、迁移、粘附及侵袭。3.HC6R1可以显著抑制小鼠肿瘤的生长并延长小鼠的生存时间。4.HC6R1可以阻断IL6介导的JAK/STAT3信号通路,通过抑制其下游STAT3蛋白的磷酸化阻滞G1期细胞周期蛋白CyclinD1和CDK4的表达从而将细胞周期抑制在G1/S期并促进细胞凋亡。
[Abstract]:Purpose 1. To identify the specificity and recognition site of HC6R1. To study the anti-tumor effect of HC6R1 on hepatocellular carcinoma and its mechanism. Methods: mice were immunized with human liver cancer and antibody was prepared by hybridoma technique. Flow cytometry and immunohistochemistry were used to detect whether HC6R1 could specifically recognize hepatoma cells and tissues. The identification sites of HC6R1 were identified by immunoprecipitation and mass spectrometry. The IL6R gene was silenced by the construction of vector transfection cells, HC6R1 and anti-IL6R were used as the first antibody, and the recognition sites were further proved by Western blotting and flow cytometry. A series of in vitro experiments, such as CCK8, scratch test, adhesion test and Transwell, were used to detect the effects of HC6R1 on the proliferation, migration, adhesion and invasion of HCC cells. Tumor models were established by subcutaneous injection of HepG2 and Huh7 cells to study the inhibitory effect of HC6R1 on tumor in vivo. Annexin V/PI double staining and flow cytometry were used to detect the effect of HC6R1 on apoptosis and cell cycle of hepatoma cells. The target genes acting on HC6R1 were detected by gene chip technique. The anti-cyclin D1 and CDK4 antibodies were used to detect the protein expression level of the target gene by Western blotting. Results 1. The preparation and identification of HC6R1. 1. Using hybridoma technique to prepare and screen a positive monoclonal antibody, HC6R1.1.2 was used to detect human hepatoma cell line / liver cancer tissue and other cells / tissues by flow cytometry and immunohistochemistry. HC6R1 can be recognized by human hepatoma cells and liver cancer tissues. However, HC6R1.1.3 could hardly be detected in normal liver cells and paracancerous tissues. It was found by immunoprecipitation and mass spectrometry that the antigen recognized by HC6R1 antibody was probably IL6R. Western blotting and flow cytometry analysis showed that the expression of HC6R1 antigen and anti-IL6R in HCC cells after knockout was significantly decreased. Further confirmed the antitumor effect of IL6R.2.HC6R1 on HCC and its mechanism 2.1 CCK8 detection showed that the proliferation rate of HC6R1 treated group was significantly slower than that of mIgG treated control group. The results of scratch test showed that HC6R1 could significantly inhibit the migration of hepatoma cells. The adhesion test and Transwell assay showed that HC6R1 could significantly inhibit the adhesion and invasion of hepatoma cells. It was found that HC6R1 could inhibit the growth of subcutaneous tumor in mice by tumorigenesis of hepatoma cells in vivo. At the same time, HC6R1 could inhibit the growth of tumor and prolong the survival time of in situ tumor-bearing mice. 2.3 by detecting apoptosis and cell cycle, HC6R1 could promote apoptosis of hepatoma cells. And can block cell cycle at G 1 / S phase, thus inhibiting the proliferation of tumor cells. Gene chip technique was used to detect the target genes acting on HC6R1. It was found that Bcl-2CX Bcl-x LHcl-x survivin D1VEFGG, HIF-1MMP 2, IL-6 and IL-10 genes were significantly activated, most of which were downstream genes of STAT3 signaling pathway. Western blotting analysis showed that HC6R1 antibody could down-regulate the expression of p-STAT3CyclinD1 and CDK4 in HepG2 and Huh7 cells. Conclusion: 1. HC6R1 can specifically recognize human hepatoma cells and tissues, and its action site is that IL6R.2.HC6R1 can significantly inhibit the proliferation and migration of HCC cells. Adhesion and invasion. 3. HC6R1 could significantly inhibit the growth of mouse tumor and prolong the survival time of mice. 4. HC6R1 could block the JAK/STAT3 signaling pathway mediated by IL6. The phosphorylation of downstream STAT3 protein inhibited the expression of cyclin CyclinD1 and CDK4 in G1 phase, thereby inhibiting cell cycle at G 1 / S phase and promoting cell apoptosis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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