本文選題：腫瘤抗原　切入點(diǎn)：食管癌　出處：《河北醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：腫瘤免疫療法作為繼手術(shù)、放化療后的抗癌新方法,其機(jī)理主要是激活機(jī)體自身生物防御機(jī)制對(duì)抗腫瘤細(xì)胞。篩選腫瘤特異性抗原是免疫治療的關(guān)鍵。癌/睪丸抗原(cancer/testis antigen,CTA)因其表達(dá)模式的特異性而成為了理想的腫瘤免疫治療靶點(diǎn)。CTA在多種腫瘤組織中具有廣泛表達(dá)的特性,而在機(jī)體正常組織中僅在睪丸等生殖相關(guān)細(xì)胞中有表達(dá),偶而在胎盤(pán)中有表達(dá),但是睪丸與胎盤(pán)等生殖相關(guān)組織是免疫豁免器官,不會(huì)表達(dá)人類白細(xì)胞抗原HLA分子(human leukocyte antigen),也不會(huì)引起機(jī)體特異性的抗原抗體免疫反應(yīng)。所以,CTA符合作為腫瘤特異性免疫治療的靶向抗原。黑色素瘤抗原基因(Melanoma-associated antigen gene,MAGE)家族屬于CTA中的一類,也表現(xiàn)為在人體正常的組織細(xì)胞中局限表達(dá)于在睪丸和胎盤(pán)等生殖相關(guān)組織細(xì)胞中,在其它正常機(jī)體組織中均不表達(dá),而在腫瘤組織中卻是高表達(dá)。鑒于MAGE家族成員不同的表達(dá)模式,MAGE可以家族分為兩個(gè)亞類,即MAGE-I型抗原和MAGE-II型抗原。MAGE-I型抗原為特異性腫瘤抗原,其還可以分為三種亞類,即MAGE-A、MAGE-B和MAGE-C。而MAGE-A中又包含12個(gè)成員,MAGE-A1~MAGE-A12。MAGE-II型抗原因其在人體所有正常組織細(xì)胞中普遍表達(dá),故不能歸屬CTA的范疇。在MAGE家族所有的成員中,MAGE-A型抗原亞家族具有更高的腫瘤特異性。在本研究中,我們利用基因轉(zhuǎn)染技術(shù)建立過(guò)表達(dá)MAGE-A9或MAGE-A11的人食管癌細(xì)胞,通過(guò)皮下種植方法構(gòu)建食管癌裸鼠荷瘤模型,測(cè)量移植瘤生長(zhǎng)過(guò)程中的瘤體積變化,繪制移植瘤生長(zhǎng)曲線,實(shí)驗(yàn)結(jié)束時(shí),處死荷瘤小鼠剝離移植瘤,并稱量瘤體重量,以分析過(guò)表達(dá)MAGE-A9或MAGE-A11基因?qū)β闶笠浦材[瘤生長(zhǎng)發(fā)展的影響,同時(shí)通過(guò)小動(dòng)物活體熒光成像系統(tǒng)檢測(cè)過(guò)表達(dá)MAGE-A9或MAGE-A11的裸鼠移植瘤組織中GFP熒光蛋白激發(fā)的熒光能量情況,旨在探討食管癌免疫治療新的靶點(diǎn)MAGE-A9與MAGE-A11在活體移植瘤中的作用。目的:探討外源性MAGE-A9和MAGE-A11基因?qū)κ彻馨┘?xì)胞裸鼠移植瘤生長(zhǎng)的影響。方法:分別將過(guò)表達(dá)MAGE-A9和MAGE-A11基因的人食管癌細(xì)胞Eca109接種到裸鼠皮下,建立裸鼠移植瘤,檢測(cè)移植瘤的生長(zhǎng)情況,再利用小動(dòng)物活體成像技術(shù)檢測(cè)移植腫瘤熒光能量值,以反映裸鼠皮下移植瘤的活性;轉(zhuǎn)染空白質(zhì)粒的細(xì)胞株接種裸鼠建立的移植瘤模型作為對(duì)照。探討MAGE-A9和MAGE-A11基因在人食管癌細(xì)胞中的作用。結(jié)果:1空載體轉(zhuǎn)染對(duì)照組、p CMV6-AC-MAGE-A9-GFP組與p CMV6-AC-MAGE-A11-GFP組,轉(zhuǎn)染成功率分別為:91.2%,92.06%,89.83%。2移植瘤最終空轉(zhuǎn)對(duì)照組有1例沒(méi)有成瘤,其他的移植瘤生長(zhǎng)呈圓形或橢圓形,個(gè)別動(dòng)物有兩個(gè)或三個(gè)分散瘤體,移植瘤總體表面光滑。3移植瘤體積生長(zhǎng)曲線顯示空轉(zhuǎn)對(duì)照組移植瘤于第15日開(kāi)始逐漸增長(zhǎng),p CMV6-AC-MAGE-A9-GFP組與p CMV6-AC-MAGE-A11-GFP組的移植瘤在第10日就出現(xiàn)明顯增長(zhǎng),整體生長(zhǎng)趨勢(shì)可見(jiàn)過(guò)表達(dá)MAGE-A9或MAGE-A11,對(duì)移植瘤生長(zhǎng)都有明顯的促進(jìn)作用(P0.01)。4空轉(zhuǎn)對(duì)照組移植瘤重量0.29±0.046g,過(guò)表達(dá)MAGE-A9或MAGE-A11的移植瘤重量0.99±0.132g和0.69±0.072g,表明過(guò)表達(dá)這兩個(gè)基因都對(duì)移植瘤生長(zhǎng)都有明顯的促進(jìn)作用(P0.01)。5空轉(zhuǎn)對(duì)照組、p CMV6-AC-MAGE-A9-GFP組、p CMV6-AC-MAGE-A11-GFP組移植瘤熒光能量值分別為37.76±2.01、69.93±6.31、67.76±7.79(CPS×105)�；铙w成像實(shí)時(shí)圖片顯示過(guò)表達(dá)MAGE-A9和MAGE-A11基因的移植瘤熒光強(qiáng)度大于空轉(zhuǎn)對(duì)照組。6與空轉(zhuǎn)組相比,兩個(gè)過(guò)表達(dá)組的MAGE-A9與MAGE-A11相對(duì)表達(dá)量(2~(-??CT))分別為3.46±0.29、6.06±0.64(P0.005)。結(jié)論:外源性MAGE-A9和MAGE-A11促進(jìn)了人食管癌細(xì)胞Eca109裸鼠移植瘤的生長(zhǎng)。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.1;R730.51

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