外源性肿瘤抗原MAGE-A9及MAGE-A11对食管癌裸鼠移植瘤生长的影响

发布时间：2018-03-17 00:14

本文选题：肿瘤抗原　切入点：食管癌　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：肿瘤免疫疗法作为继手术、放化疗后的抗癌新方法,其机理主要是激活机体自身生物防御机制对抗肿瘤细胞。筛选肿瘤特异性抗原是免疫治疗的关键。癌/睾丸抗原(cancer/testis antigen,CTA)因其表达模式的特异性而成为了理想的肿瘤免疫治疗靶点。CTA在多种肿瘤组织中具有广泛表达的特性,而在机体正常组织中仅在睾丸等生殖相关细胞中有表达,偶而在胎盘中有表达,但是睾丸与胎盘等生殖相关组织是免疫豁免器官,不会表达人类白细胞抗原HLA分子(human leukocyte antigen),也不会引起机体特异性的抗原抗体免疫反应。所以,CTA符合作为肿瘤特异性免疫治疗的靶向抗原。黑色素瘤抗原基因(Melanoma-associated antigen gene,MAGE)家族属于CTA中的一类,也表现为在人体正常的组织细胞中局限表达于在睾丸和胎盘等生殖相关组织细胞中,在其它正常机体组织中均不表达,而在肿瘤组织中却是高表达。鉴于MAGE家族成员不同的表达模式,MAGE可以家族分为两个亚类,即MAGE-I型抗原和MAGE-II型抗原。MAGE-I型抗原为特异性肿瘤抗原,其还可以分为三种亚类,即MAGE-A、MAGE-B和MAGE-C。而MAGE-A中又包含12个成员,MAGE-A1~MAGE-A12。MAGE-II型抗原因其在人体所有正常组织细胞中普遍表达,故不能归属CTA的范畴。在MAGE家族所有的成员中,MAGE-A型抗原亚家族具有更高的肿瘤特异性。在本研究中,我们利用基因转染技术建立过表达MAGE-A9或MAGE-A11的人食管癌细胞,通过皮下种植方法构建食管癌裸鼠荷瘤模型,测量移植瘤生长过程中的瘤体积变化,绘制移植瘤生长曲线,实验结束时,处死荷瘤小鼠剥离移植瘤,并称量瘤体重量,以分析过表达MAGE-A9或MAGE-A11基因对裸鼠移植肿瘤生长发展的影响,同时通过小动物活体荧光成像系统检测过表达MAGE-A9或MAGE-A11的裸鼠移植瘤组织中GFP荧光蛋白激发的荧光能量情况,旨在探讨食管癌免疫治疗新的靶点MAGE-A9与MAGE-A11在活体移植瘤中的作用。目的:探讨外源性MAGE-A9和MAGE-A11基因对食管癌细胞裸鼠移植瘤生长的影响。方法:分别将过表达MAGE-A9和MAGE-A11基因的人食管癌细胞Eca109接种到裸鼠皮下,建立裸鼠移植瘤,检测移植瘤的生长情况,再利用小动物活体成像技术检测移植肿瘤荧光能量值,以反映裸鼠皮下移植瘤的活性;转染空白质粒的细胞株接种裸鼠建立的移植瘤模型作为对照。探讨MAGE-A9和MAGE-A11基因在人食管癌细胞中的作用。结果:1空载体转染对照组、p CMV6-AC-MAGE-A9-GFP组与p CMV6-AC-MAGE-A11-GFP组,转染成功率分别为:91.2%,92.06%,89.83%。2移植瘤最终空转对照组有1例没有成瘤,其他的移植瘤生长呈圆形或椭圆形,个别动物有两个或三个分散瘤体,移植瘤总体表面光滑。3移植瘤体积生长曲线显示空转对照组移植瘤于第15日开始逐渐增长,p CMV6-AC-MAGE-A9-GFP组与p CMV6-AC-MAGE-A11-GFP组的移植瘤在第10日就出现明显增长,整体生长趋势可见过表达MAGE-A9或MAGE-A11,对移植瘤生长都有明显的促进作用(P0.01)。4空转对照组移植瘤重量0.29±0.046g,过表达MAGE-A9或MAGE-A11的移植瘤重量0.99±0.132g和0.69±0.072g,表明过表达这两个基因都对移植瘤生长都有明显的促进作用(P0.01)。5空转对照组、p CMV6-AC-MAGE-A9-GFP组、p CMV6-AC-MAGE-A11-GFP组移植瘤荧光能量值分别为37.76±2.01、69.93±6.31、67.76±7.79(CPS×105)。活体成像实时图片显示过表达MAGE-A9和MAGE-A11基因的移植瘤荧光强度大于空转对照组。6与空转组相比,两个过表达组的MAGE-A9与MAGE-A11相对表达量(2~(-??CT))分别为3.46±0.29、6.06±0.64(P0.005)。结论:外源性MAGE-A9和MAGE-A11促进了人食管癌细胞Eca109裸鼠移植瘤的生长。
[Abstract]:Tumor immunotherapy as following surgery, a new method of anticancer chemotherapy, its mechanism is to activate the body's own biological defense mechanism against tumor cells. Screening of tumor specific antigen is the key for immunotherapy of cancer / testis antigens (cancer/testis, antigen, CTA) for specific expression patterns and become the ideal tumor immunity the treatment targets of.CTA has the characteristics of widely expressed in tumor tissues and normal tissues in the body only in the testis and other reproductive cells, occasionally in placenta expressed in the testis and placenta, but reproductive tissue is related to immune privilege organ, not the expression of human leukocyte antigen HLA (human leukocyte antigen), will not cause an immune response to antigen antibody specificity. Therefore, CTA qualifies as a tumor specific immunotherapy targeting antigen. Gene anti melanoma (Mela Noma-associated antigen gene, MAGE) family belongs to a class of CTA, also expressed in human normal tissues expressed in testis and placenta in the limitations of other reproductive tissues, in other normal tissues were not expressed, but high expression in tumor tissue. Expression of MAGE family members in different mode the MAGE family can be divided into two subgroups, namely MAGE-I type MAGE-II type.MAGE-I type antigen and antigen antigen specific tumor antigen, which can also be divided into three sub categories, namely MAGE-A, MAGE-B and MAGE-C. in MAGE-A, and consists of 12 members, MAGE-A1~MAGE-A12.MAGE-II anti reason is generally expressed in all of the human body the normal tissue cells, so it can belong to the category of CTA. All of the members in the MAGE family, MAGE-A antigen subfamily has a higher tumor specificity. In this study, we used gene transfection technology The establishment of over expression of MAGE-A9 or MAGE-A11 in human esophageal cancer cells, construct the tumor model of esophageal carcinoma in nude mice by subcutaneous implantation method, the tumor volume changes during the growth of tumors were measured in the draw the tumor growth curve, at the end of the experiment, were stripped of tumor bearing mice transplanted tumor, and weighing tumor weight, in order to analyze the effects of over expression the MAGE-A9 or MAGE-A11 gene on the growth of transplanted tumor development, at the same time by small animal in vivo fluorescence imaging system for detection of fluorescence energy of GFP fluorescent protein induced expression of MAGE-A9 or MAGE-A11 in the tumor tissues, to investigate esophageal cancer immunotherapy targets MAGE-A9 and MAGE-A11 new in vivo transplantation tumor in vitro. Objective: To investigate the effects of exogenous MAGE-A9 and MAGE-A11 gene on the growth of esophageal carcinoma cells in nude mice. Methods: the expression of MAGE-A9 and MAGE-A11 gene were non carcinoma Eca109 cells were inoculated subcutaneously into nude mice to establish the xenograft tumor growth, tumor detection, then detected by in vivo imaging technology of small animal transplanted tumor fluorescence energy value to reflect the activity of nude mice transfected with blank plasmid; cell lines established in nude mice xenograft model as control. The effects of MAGE-A9 and MAGE-A11 gene in human esophageal cancer cells. Results: 1 empty plasmid control group, P CMV6-AC-MAGE-A9-GFP group and P CMV6-AC-MAGE-A11-GFP group, the success rate of transfection were 91.2%, 92.06%, 89.83%.2 tumor eventually idling 1 cases in the control group without tumor, other xenografts were round or oval, there are two individual animal or three separate tumor xenografts, overall smooth surface.3 tumor volume growth curve showed that idling control group transplanted in fifteenth began to gradually increase, P CMV6-AC-MAGE-A9-GFP Group P and group CMV6-AC-MAGE-A11-GFP on transplanted tumor grew significantly in tenth, the overall growth trend can be seen the expression of MAGE-A9 or MAGE-A11, has obvious effect on promoting the growth of tumor control group (P0.01).4 idle tumor weight 0.29 + 0.046g, over expression of tumor weight of MAGE-A9 or MAGE-A11 0.99 + 0.132g and 0.69 + 0.072g, showed that over expression of these two genes on the growth of transplanted tumor were significantly promoted (P0.01).5 idling control group, P CMV6-AC-MAGE-A9-GFP group, P CMV6-AC-MAGE-A11-GFP group transplanted fluorescence energy values were 37.76 + 2.01,69.93 + 6.31,67.76 + 7.79 (CPS * 105). Real time image displayed in vivo imaging of transplanted tumor the fluorescence intensity of the expression of MAGE-A9 and MAGE-A11 gene of.6 compared with the control group is greater than the idle idle group, two over expression of relative expression of MAGE-A9 and MAGE-A11 group (2~ (?? CT)) were 3.46 + 0.29,6.06 0.64 (P0.005). Conclusion: exogenous MAGE-A9 and MAGE-A11 promote the growth of human esophageal cancer cells in Eca109 nude mice.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1;R730.51

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