Background and objective: The absence of α-gal in humans is caused by the inactivity of α-1,3GT gene. However, humans have pre-existing and abundant anti-gal antibodies. Xenotransplantation procedures have indicated the high potential of introducing α-1,3GT gene to synthesize α-gal for cancer gene therapy by mimicking hyper-acute rejection. The aim of this study is to construct a lung cancer A549 cell line that expressed α-gal, and to observe the antitumor mechanisms mediated by human serum.
Methods: A549 cells 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 cells, including morphology and proliferation, were examined. α-1,3GT mRNA expression was detected by RT-PCR. Direct immunofluorescence staining and flow cytometry (FCM) were used to analyze the synthesis of α-gal in A549-GT. The binding of human serum IgM and C3 with A549-GT were also detected.
Results: α-1,3GT mRNA was expressed in A549-GT. Direct immunofluorescence staining and FCM indicated a high and stable α-gal expression rate in A549-GT. Compared with parental A549 cells, the biological characteristics of A549-GT were unaltered. α-Gal expression was not detected in the human fetal lung fibroblast cell line MRC-5 even though A549-GT and its culture medium were cultivated with the enzyme. Immunofluorescence staining and FCM also indicated abundant binding between A549-GT treated with human serum and IgM/C3.
Conclusions: α-Gal expression in tumor cells by gene transduction can induce complement-dependent cytototic antitumor effects.
背景与目的: 人体α-1, 3半乳糖基转移酶（α-1, 3Galactosyltransferase, 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-IB4 lectin）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, 3GT mRNA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。