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
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摘要
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p LVX-CD-IRES-Zs Green1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。
Objective To research on the glioma therapy based on the established human glioma cell carrying CD gene. Methods CD gene was inserted to the p LVX- IRES- Zs Green1 lentivirus vector using the In- fusion gene clone method. The lentivirus was packaged and its titer was detected in 293 T cells. The glioma cells carrying CD gene were obtained by infected them with the p LVX- CD-IRES- Zs Green1 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 p LVX- CD- IRES- Zs Green1 was successfully constructed and the lentivirus was packaged in vitro in 293 T 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 prodrug5- 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.
Objective To research on the glioma therapy based on the established human glioma cell carrying CD gene. Methods CD gene was inserted to the p LVX- IRES- Zs Green1 lentivirus vector using the In- fusion gene clone method. The lentivirus was packaged and its titer was detected in 293 T cells. The glioma cells carrying CD gene were obtained by infected them with the p LVX- CD-IRES- Zs Green1 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 p LVX- CD- IRES- Zs Green1 was successfully constructed and the lentivirus was packaged in vitro in 293 T 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 prodrug5- 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.
引文
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2 Chen J,Mckay RM,Parada LF.Malignant glioma:lessons from genomics,mouse models,and stem cells[J].Cell,2012,149(1):36-47
3 Murphy AM,Rabkin SD.Current status of gene therapy for brain tumors[J].Translat Res,2013,161(4):339-354
4 Duarte S,Carle G,Faneca H,et al.Suicide gene therapy in cancer:where do we stand now?[J].Cancer Lett,2012,324(2):160-170
5 Ostertag D,Amundson KK,Lopez EF,et al.Brain tumor eradication and prolonged survival from intratumoral conversion of 5-fluorocytosine to 5-fluorouracil using a nonlytic retroviral replicating vector[J].Neuro-Oncol,2012,14(2):145-159
6 Jin G,Zhou Y,Chai Q,et al.VP22 and cytosine deaminase fusion gene modified tissue-engineered neural stem cells for glioma therapy[J].J Cancer Res Clin Oncol,2013,139(3):475-483
7 Wakimoto H,Kesari S,Farrell C J,et al.Human glioblastoma-derived cancer stem cells:establishment of invasive glioma models and treatment with oncolytic herpes simplex virus vectors[J].Cancer Res,2009,69(8):3472-3481
8 Zhu G,Su W,Jin G,et al.Glioma stem cells targeted by oncolytic virus carrying endostatin-angiostatin fusion gene and the expression of its exogenous gene in vitro[J].Brain Res,2011,1390:59-69
9 Zhang GB,Jin GS,Nie XT,et al.Enhanced antitumor efficacy of an oncolytic herpes simplex virus expressing an endostatin-angiostatin fusion gene in human glioblastomas stem cell xenografts[J].PLo S One,2014,9(4):e95872