CEA-EPO质粒的构建及诱导造血干细胞定向生成红细胞疫苗抗结肠癌的研究

Abstract

目的：研究CEA-EPO质粒的构建及诱导造血干细胞定向生成红细胞疫苗抗结肠癌的研究。方法：将EPO-cDNA插入pcDNA3.1质粒的多克隆位点，用CEA基因启动子替代pcDNA3.1质粒的CMV启动子，构建成CEA-EPO重组质粒。用含抗体CD117磁珠分离纯化和收集小鼠骨髓造血干细胞，用重组腺病毒法将CEA-EPO基因体外转染CEA-EPOmRNA基因到造血干细胞内，检测CEA-EPO基因和蛋白表达，将疫苗注入小鼠体内，进行扩增，使造血干细胞定向生成携带CEA-EPO双基因红细胞，经过提取和纯化获得CEA-EPO基因共转染红细胞疫苗，再进行双基因共转染红细胞疫苗的体外和体内实验。结果：成功收集造血干细胞，流式细胞仪分析结果表明：纯化前后有明显差异(P < 0.05)；检测CEA-EPOmRNA和蛋白表达的结果表明：造血干细胞体外转染CEA-EPOmRNA基因成功，获得CEA-EPO双基因共转染红细胞疫苗；应用细胞红系相关标志抗体GPA和CD71标记，利用流式细胞证实红细胞疫苗培养成功；研究相关疫苗激活的单核细胞对结肠癌细胞株CT26细胞杀伤作用表明：在比例为40:1时，CEA-EPO疫苗组杀伤作用为57.34 ± 1.13，高于空载组、CEA疫苗组和EPO疫苗组(P < 0.05)，体内试验的成瘤性研究表明：CEA-EPO疫苗组肿瘤的生长的大小为0.29 ± 0.11 (cm3)；高于其他各组的大小(P < 0.05)，CEA-EPO疫苗组的成活期分别为：69 ± 6.91天，高于其他各组的大小(P < 0.05)；CEA-EPO疫苗组在肿瘤大小和小鼠的生存时间上，与空载组、CEA疫苗组，EPO疫苗组之间差异有统计学意义(P < 0.05)。结论：携带CEA-EPO双基因肿瘤抗原的红细胞疫苗对结肠癌有明显的抗肿瘤作用，既可以诱导造血干细胞定向分化成红细胞疫苗，又可以合成肿瘤抗原的红细胞疫苗，是临床治疗结肠的重要疫苗。 Objective: To investigate the effects of recombinant adenovirus-mediated co-transfection of CEA gene and EPO gene on promoting hematopoietic stem cell directly producing erythrocyte vaccine against colon cancer. Method: The expression adenovirus vectors carrying CEA and EPO or green fluorescent protein (GFP) gene were constructed respectively, and recombinant adenovirus car-rying CEA, EPO or GFP were packaged and produced respectively. The bone marrow-derived MSCs of mice were isolated and cultured in vitro by anti-CD117magnetic bead separation, and were transfected with CEA (CEA group), EPO (EPO group) or GFP(GFP group), co-transfected with CEA and EPO (CEA and EPO group), or transfected with no virus (controlgroup). The expression of CEA and EPO gene and protein after transfection in supernatant fluid of culture was detected by RT-PCR and western blot in each group. We have checked and obtained the vaccine with co-transfection of CEA gene and EPO gene by cell red line marker antibody CD71 and GPA, then we carried on experiments with the vaccine in vitro and in vivo. Result: We have successfully gathered the hematopoietic stem cells; flow cytometry analysis result showed that there were significant differences before and after purification (P < 0.05). The expression of double genes (CEA-EPO gene) and protein shows CEA-EPO gene were successfully transfected into the hematopoietic stem cells. We have confirmed erythrocyte vaccine with co-transfection of CEA and EPO gene by antibody CD71 and GPA with flow cytometry. The monocytes cytotoxicity on colon cancer cell line CT26 showed that CEA-EPO group was 57.34 ± 1.13% in proportion of 40:1. Compared with CEAg group and EPO group, there was significant difference between them (P < 0.05). In the experimentation of neoplasma format, the size of murine growth of tumor was 0.29 ± 0.11 cm3 differently in the group of CEA-EPO group; the number of murine live time on day was 69 ± 6.91 days. Compared with the group of CEA and plasmid, EPO, the group of CEA-EPO was significant differences in size and live time (P < 0.05). Conclusion: The erythrocyte vaccine with co-transfection of CEA gene and EPO gene has efficient anti-tumor effects on colon cancer. It not only can promote hematopoietic stem cell directly producing erythrocyte vaccine, but also can produce tumor Antigen vaccine against stomach cancer.