CEA-EPO 质粒的构建及其诱导造血干细胞定向生成的红细胞疫苗治疗结肠癌的研究


目的 研究癌胚抗原(CEA)-红细胞生成素(EPO)质粒的构建及诱导造血干细胞定向生成红细胞疫苗的抗结肠癌效果。 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 质粒的疱疹病毒(CMV)启动子,构建成 CEA-EPO 重组质粒(同时构建空载体质粒、CEA 质粒和 EPO 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 基因体外转染到造血干细胞内,检测 CEA-EPO 基因及其蛋白的表达;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 基因的红细胞,经过提取和纯化获得 CEA-EPO 基因共转染红细胞疫苗,再进行基因共转染红细胞疫苗的体外和体内实验。实验分组为:空载体组、CEA 组、EPO 组和 CEA-EPO 组。 结果 成功收集造血干细胞,流式细胞仪分析结果表明:分选后的阳性管标本纯化后的 CD117 阳性细胞纯度较纯化前升高,差异有统计学意义(P=0.001);CEA-EPO mRNA 及其蛋白表达的检测结果表明:造血干细胞体外转染 CEA-EPO mRNA 基因成功,获得 CEA-EPO 基因共转染红细胞疫苗;采用细胞红系相关标志抗体 GPA 和 CD71 标记,并利用流式细胞仪证实红细胞疫苗培养成功;相关疫苗激活的单核细胞对结肠癌细胞株 CT26 细胞杀伤作用实验结果表明:在比例为 40∶1 时,CEA-EPO 组的杀伤作用强于空载体组、CEA 组和 EPO 组(P<0.05);体内试验的成瘤性研究结果表明:处理后 2 周 CEA-EPO 组的肿瘤体积大于其他 3 组(P<0.05),且 CEA-EPO 组的生存时间长于其他 3 组(P<0.05)。 结论 携带 CEA-EPO 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,又可以合成携带肿瘤抗原的红细胞疫苗,有望发展成为临床治疗结肠癌的重要疫苗。

Objective To investigate the effects of recombinant adenovirus-mediated co-transfection of carcinoembryonic antigen (CEA) gene and erythropoietin (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 carrying CEA, EPO or GFP were packaged and produced respectively. The bone marrow-derived mesenchymal stem cells (MSCs) of mice were isolated and cultured in vitro by anti-CD117 magnetic bead separation, and were transfected with CEA (CEA group), EPO (EPO group) or GFP (GFP group), co-transfected with CEA and EPO (CEA-EPO group), or transfected with no virus (control group). The expression of CEA and EPO gene and its protein after transfection in supernatant fluid of culture was detected by RT-PCR and Western blot in each group. We had 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 had 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 had 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 ? of CEA-EPO group was higher than other 3 groups when in proportion of 40∶1 (P<0.05). In the experimentation of neoplasma format, the survival time of CEA-EPO group was higher than other 3 groups too (P<0.05). Conclusions The erythrocyte vaccine with co-transfection of CEA gene and EPO gene has efficient anti-tumor effects on colon cancer. Not only can promote hematopoietic stem cell directly producing erythrocyte vaccine, but also can produce tumor antigen vaccine against colon cancer.

关键词: 结肠癌; 癌胚抗原; 红细胞生成素; 质粒; 转染; 红细胞疫苗

Key words: colon cancer; carcinoembryonic antigen; erythropoietin; plasmid; transfection; erythrocyte vaccine

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