人胶质瘤自杀基因细胞系的建立及其在胶质瘤治疗研究中的应用

目的 构建CD基因工程化人脑胶质瘤细胞并在此基础上进行胶质瘤治疗研究。方法 通过In-fusion基因克隆的方法,构建携带CD基因慢病毒载体;通过293T细胞体外包装获得慢病毒,并测定病毒效价;通过慢病毒体外感染细胞获得CD基因工程化人脑胶质瘤细胞;通过流式分选获得基因稳定表达细胞;通过CCK-8评价加入5-FC前药和(或)HSV-1后细胞存活率。结果 成功构建pLVX-CD-IRES-ZsGreen1慢病毒载体并体外包装获得相应慢病毒;慢病毒感染人脑胶质瘤U87细胞后获得携带CD基因的阳性细胞,并通过体外培养30天后流式分选获得稳定阳性细胞;加入前药5-FC和(或)HSV-1后对携带CD基因的U87细胞杀伤作用明显(P<0.01),其中5-FC联合HSV-1对胶质瘤细胞作用更加高效(P<0.01)。结论 成功获得携带CD基因的人脑胶质瘤细胞;前药5-FC联合HSV-1对胶质瘤细胞具有更加高效的杀伤疗效。

Therapeutic Effect of the Established Glioma Cells Carrying Cytosine Deaminase Suicide Gene

Objective To research on the glioma therapy based on the established human glioma cell carrying CD gene. Methods CD gene was inserted to the pLVX-IRES-ZsGreen1 lentivirus vector using the In-fusion gene clone method. The lentivirus was packaged and its titer was detected in 293T cells. The glioma cells carrying CD gene were obtained by infected them with the pLVX-CD-IRES-ZsGreen1 lentivirus following the fluorescence activated cell sorting (FACS). The cell survival rate was evaluated when adding the prodrug 5-FC or/and HSV-1 using the CCK-8 method. Results The lentivirus vector pLVX-CD-IRES-ZsGreen1 was successfully constructed and the lentivirus was packaged in vitro in 293T cells and then infected the U87 cells. The U87 cells carrying CD gene was successfully obtained following FACS after cultured in vitro for 30 days. The inhibition effect was significant when adding the prodrug 5-FC or/and HSV-1 to the U87-CD cells(P<0.01). And the cytotoxic effect of combined usage of 5-FC and HSV-1 was significantly enhanced compared to usage of 5-FC or HSV-1 alone(P<0.01). Conclusion We successfully obtained the glioma cells carrying CD gene. And the efficacy was enhanced when combined use of the prodrug 5-FC and the oncolytic virus HSV-1 in the glioma cells carrying CD gene.