单ADP核糖基转移酶ARTD10调控大肠癌细胞增殖及其机制的初步研究

发布时间：2018-06-22 11:03

本文选题：ARTD10 + 大肠癌　；参考：《重庆医科大学》2017年硕士论文

【摘要】：背景及目的:ADP核糖基化是一种重要的翻译后修饰,可参与多种生理和病理行为的调控,如细胞死亡、细胞周期和DNA修复。我们之前的研究发现单ADP核糖基化转移酶1(clostridial toxin-like ADP-ribosyltransferases 1,ARTC1)与大肠癌的增殖、凋亡、迁移、转移、血管形成以及其他生物学行为密切相关。单ADP核糖基转移酶ARTD10(ADP-ribosyltransferase diphtheria toxin-like 10)是新近发现的一种细胞内单ADP核糖基化转移酶,其与肿瘤性疾病的关系研究甚少。ARTD10在大肠癌组织中的表达以及其对大肠癌生物学行为的影响尚不清楚。本课题首先检测ARTD10在人大肠癌组织中及癌旁正常大肠粘膜中的表达情况,探讨ARTD10在人大肠癌组织中及癌旁正常大肠粘膜中的表达差异;并以小鼠大肠癌CT26细胞系为研究对象,通过在体和离体实验,观察ARTD10对大肠癌CT26细胞增殖的影响,并初步探讨其可能的机制,为ARTD10作为大肠癌靶向治疗的潜在靶点提供初步实验依据。方法:1.免疫组化方法检测51例不同分化程度的人大肠癌组织及癌旁正常大肠黏膜中ARTD10的表达,激光共聚焦显微镜观察人大肠癌细胞系LOVO细胞、SW480细胞及小鼠大肠癌细胞系CT26细胞中ARTD10的表达情况。2.以高表达ARTD10的小鼠大肠癌细胞系CT26细胞为研究对象,使用ARTD10特异性抑制剂处理后,以CCK-8法检测CT26细胞增殖能力变化。构建Balb/c小鼠腋窝皮下移植瘤模型,观察ARTD10对CT26细胞移植瘤生长的影响。3.使用Western Blot方法检测ARTD10特异性抑制剂处理后CT26细胞核转位因子β-catenin的表达和功能水平及其下游信号蛋白AXIN2和c-MYB的蛋白表达水平变化。结果:1.免疫组化结果显示,ARTD10在人大肠癌组织和癌旁正常大肠黏膜中都有表达,主要定位于胞质,偶可见于胞核;人大肠癌组织中ARTD10的阳性率(85%)明显高于癌旁正常大肠黏膜组织(19.4%)(P㩳0.05);不同分级的大肠癌中ARTD10的阳性程度有显著性差异(P㩳0.05),ARTD10的阳性程度与大肠癌的分级呈正相关(P㩳0.05),但在不同年龄、性别、肿瘤部位、淋巴结转移、远处转移、肿瘤分期和浸润深度的大肠癌中ARTD10的表达没有显著差异(P0.05)。2.激光共聚焦显微镜观察结果显示,ARTD10在分化较高的人大肠癌SW480细胞中表达水平较低,而在分化较差的小鼠大肠癌CT26细胞系和人大肠癌LOVO细胞系中表达水平明显高于前者。3.CCK8结果显示,使用ARTD10特异性抑制剂抑制CT26细胞内ARTD10酶活性后,CT26细胞的增殖明显受到抑制;并且随着抑制剂浓度的增加,抑制作用也增强,表现出明显的浓度依赖性(P0.05),在ARTD10特异性抑制剂浓度为0.5μM时对CT26细胞增殖抑制作用最明显。用0.5μM浓度的ARTD10特异性抑制剂处理CT26细胞24h,48h,72h,96h和120h后,处理时间为72h时对CT26细胞增殖抑制作用最强。4.当腹腔注射ARTD10特异性抑制剂(2mg/kg)后,Balb/c小鼠腋窝皮下移植瘤重量和体积均明显低于对照组(P㩳0.05)。5.Western Blot结果显示,使用特异性抑制剂抑制ARTD10酶活性后,CT26细胞全细胞和细胞核的β-catenin蛋白表达水平较对照组都明显降低(P0.05);β-catenin下游蛋白AXIN2表达增高(P0.05),而c-MYB表达降低(P0.05)。在Balb/c小鼠腋窝皮下移植瘤中,当ARTD10被特异性抑制剂抑制,移植瘤全细胞和移植瘤细胞核中的β-catenin蛋白表达水平也较对照组显著降低(P0.05)。结论:1.ARTD10在大肠癌组织中的表达水平明显高于癌旁正常的大肠黏膜组织,ARTD10的阳性程度与肿瘤的恶性程度呈正相关。2.ARTD10可促进大肠癌CT26细胞的增殖。3.ARTD10促进大肠癌CT26细胞的增殖,可能与ARTD10调节核转位蛋白β-catenin的表达和活性,进一步影响β-catenin下游增殖相关蛋白c-MYB、AXIN2的表达有关。
[Abstract]:Background and purpose: ADP ribonylation is an important post-translational modification that can participate in the regulation of a variety of physiological and pathological behaviors, such as cell death, cell cycle and DNA repair. Our previous study found that single ADP ribonucleotransferase 1 (clostridial toxin-like ADP-ribosyltransferases 1, ARTC1) and colorectal cancer proliferation, apoptosis, migration, Metastasis, angiogenesis and other biological behavior are closely related. Single ADP ribonucleotransferase ARTD10 (ADP-ribosyltransferase diphtheria toxin-like 10) is a newly discovered intracellular single ADP ribonucleotransferase, and its relationship with tumor disease is less than the expression of.ARTD10 in colorectal carcinoma and its effect on colorectal cancer The effect of biological behavior is not clear. Firstly, the expression of ARTD10 in human colorectal carcinoma tissue and normal colorectal mucosa near cancer was first detected, and the difference in expression of ARTD10 in human colorectal carcinoma tissues and normal colorectal mucosa adjacent to cancer was investigated. The CT26 cell line of large intestine cancer in mice was used as the research object, and the observation was carried out in vivo and in vitro experiments. The effect of ARTD10 on the proliferation of colorectal cancer CT26 cells was investigated and its possible mechanism was preliminarily explored to provide a preliminary experimental basis for the potential target of ARTD10 as a target therapy for colorectal cancer. Method: 1. immunohistochemical method was used to detect the expression of ARTD10 in the colorectal cancer tissues and the normal colorectal mucosa adjacent to the cancer of different degrees of differentiation, and the laser copolymerization was shown. The expression of ARTD10 in human colorectal cancer cell line LOVO cells, SW480 cells and mouse colorectal cancer cell line CT26 cells was observed by microscope.2...2. was used to study the CT26 cells of colorectal cancer cell lines with high expression of ARTD10 in mice. The proliferation ability of CT26 cells was detected by CCK-8 method. The axillary growth of Balb/c mice was constructed by the CCK-8 method. The effect of ARTD10 on the growth of CT26 cell xenografts,.3. using Western Blot method to detect the expression and function level of CT26 nuclear transposition factor beta -catenin, and the changes in the protein expression level of the downstream signal protein AXIN2 and c-MYB. Results: 1. immunohistochemical results showed, A RTD10 was expressed in the human colorectal carcinoma tissue and the normal colorectal mucosa adjacent to the carcinoma, which was mainly located in the cytoplasm and even in the nucleus. The positive rate of ARTD10 in the colorectal cancer tissue was significantly higher than that of the normal colorectal mucosa (19.4%) (P? 0.05) beside the carcinoma (P? 0.05); the positive degree of ARTD10 in different classification of colorectal cancer was significantly different (P? 0.05), and the positive of ARTD10 The degree of sex was positively correlated with the classification of colorectal cancer (P? 0.05), but there was no significant difference in the expression of ARTD10 at different age, sex, tumor site, lymph node metastasis, distant metastasis, tumor staging and infiltration depth (P0.05).2. laser confocal microscope observation results showed that ARTD10 was in the SW480 cells with higher differentiation of human colorectal cancer cells. The expression level of the CT26 cell line and the human colorectal cancer LOVO cell line in the poorly differentiated mice was significantly higher than that of the former.3.CCK8. The proliferation of CT26 cells was obviously inhibited after the use of ARTD10 specific inhibitors to inhibit the ARTD10 activity in CT26 cells, and the inhibition was inhibited with the increase of the inhibitor concentration. The effect was also enhanced, showing a significant concentration dependence (P0.05), the most obvious inhibitory effect on the proliferation of CT26 cells when the concentration of ARTD10 specific inhibitors was 0.5 M. ARTD10 specific inhibitors of ARTD10 concentration were used to treat CT26 cell 24h, 48h, 72h, 96h and 120h. After the injection of ARTD10 specific inhibitor (2mg/kg), the weight and volume of subcutaneous transplantation tumor in Balb/c mice were significantly lower than that of the control group (P? 0.05).5.Western Blot results showed that the expression level of beta -catenin protein in whole cells and nuclei of CT26 cells was significantly lower than that of the control group (P0.05) after the inhibition of ARTD10 enzyme activity by specific inhibitors (P0.05). The expression of catenin downstream protein AXIN2 increased (P0.05) and c-MYB expression decreased (P0.05). In the subcutaneous tumor of axillary subcutaneous transplantation in Balb/c mice, when ARTD10 was suppressed by specific inhibitors, the expression level of beta -catenin in the whole cell and transplanted cell nucleus of the transplanted tumor was also significantly lower than that of the control group (P0.05). Conclusion: 1.ARTD10 in colorectal cancer tissue The level of ARTD10 was significantly higher than that of the normal colorectal mucosa, and the positive degree of the tumor was positively correlated with the malignant degree of the tumor..2.ARTD10 could promote the proliferation of colorectal cancer CT26 cells and promote the proliferation of CT26 cells in colorectal cancer. It may regulate the expression and activity of the nuclear transposition protein beta -catenin with ARTD10, and further influence the downstream of the beta -catenin. The expression of proliferation related protein c-MYB, AXIN2 is related.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

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