异种移植抗原α-gal介导人血清杀伤A549细胞的实验研究  

Experimental Study on A549 Cell Death Mediated by Xenoantigen α-galin Human Serum

作　　者：朱圣明[1] 谢玲[2] 郑鸿[3] 秦凤[3] 刘美[3] 骆志国[1] 王艳萍[3,3] 

机构地区：[1]湖北医药学院附属太和医院肿瘤科,十堰442000 [2]湖北医药学院附属太和医院皮肤科,十堰442000 [3]四川大学华西医院肿瘤分子诊断研究室,成都610041

出　　处：《中国肺癌杂志》2012年第11期630-637,共8页Chinese Journal of Lung Cancer

基　　金：国家自然科学基金项目(No.30470762)资助~~

摘　　要：背景与目的人体α-1,3半乳糖基转移酶(α-1,3Galactosy ltransferase,α-1,3GT)基因功能失活而不表达α-半乳糖基(α-galactosyl,α-gal)表位,但天然存在着大量抗α-gal抗体,异种器官移植研究结果提示,在人肿瘤细胞上重新表达异种移植抗原α-gal,可能诱发类似于宿主抗移植物超急性排斥反应的抗肿瘤效应。本研究通过基因导入手段,建立稳定表达异种移植抗原α-gal的转基因人肺癌细胞系,探讨α-gal介导的人血清抗肿瘤的可能性及其机制。方法将前期成功构建的α-1,3GT基因真核表达质粒pEGFP-N1-GT瞬时转染人肺腺癌细胞A549,筛选并建立稳定的转基因细胞系A549-GT。MTT增殖实验和显微镜观察转基因细胞生物学特性变化;RT-PCR检测A549-GT中α-1,3GT基因mRNA表达;荧光素标记的凝集素(FITC-BS-IB4lectin)染色检测α-1,3GT在肿瘤细胞表面合成α-gal的能力;A549-GT细胞及其培养基分别与正常人细胞共培养,检验α-1,3GT基因的稳定性和酶活性的稳定性;人血清结合实验检测A549-GT与IgM和补体C3结合情况。结果RT-PCR检测到转基因细胞系A549-GT中有α-1,3GTmRNA表达。荧光显微镜和流式细胞术检测结果显示:A549-GT能够长期稳定表达异种移植抗原α-gal表位;A549-GT与其亲本细胞在生长形态及增殖速度上无明显差异;A549-GT细胞及其培养基分别与正常人胚肺成纤维细胞MRC-5共培养均不能使MRC-5获得α-gal合成能力;经人血清处理后,荧光免疫实验观察到转基因细胞系A549-GT能与血清抗体IgM结合并诱导补体C3结合。结论异种移植抗原α-gal在肿瘤细胞上的重新表达,可能通过补体依赖的细胞毒机制,介导类似于异种器官移植排斥反应的抗肿瘤效应。Background and objective The absence of a-gal in humans is caused by the inactivity of a-1,3GT gene. However, humans have preexisting and abundant anti-gal antibodies. Xenotransplantation procedures have indicated the high potential of introducing a-l,3GT gene to synthesize a-gal for cancer gene therapy by mimicking hyperacute rejection. The aim of this study is to construct a lung cancer A549 cell line that expressed a-gal, and to observe the antitumor mechanisms mediated by human serum. Methods A549 ceils were transfected with pEGFP-N1-GT plasmids constructed in a previous study. A stable transgenic cell line, A549-GT, was then selected and cultivated. The biological characteristics of A549-GT cellsj including morphology and proliferation, were examined, a-1,3GT mRNA expression was detected by RT- PCR. Direct immunotluorescence staining and flow cytometry （FCM） were used to analyze the synthesis of a-gal in AS49- GT. The binding of human serum IgM and C3 with A549-GT were also detected. Results a-1,3GT mRNA was expressed in A549-GT. Direct immunofluorescence staining and FCM indicated a high and stable a-gal expression rate in A549-GT. Compared with parental A549 ceils, the biological characteristics of AS49-GT were unaltered, a-Gal expression was not detected in the human fetal lung fibroblast cell line MKC-5 even though A549-GT and its culture medium were cultivated with the enzyme. Immunofluorescence staining and FCM also indicated abundant binding between AS49-GT treated withhuman serum and IgM/C3. Conclusion a-Gal expression in tumor cells by gene transduction can induce complementdependent cytototic antitumor effects.

关 键 词：α1 3-GT基因 α-半乳糖基 异种抗原 A549细胞 

分 类 号：R73-3[医药卫生—肿瘤]