摘要
AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC(50)) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean +/- SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 X 10(14)pfu/L(-1) after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC(50) values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were 】15000, 216.5+/-38.1 and 128.8+/-25.4 micromol.L(-1), P【0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the m.o.i of adenoviruses were enhanced (The value of IC(50) of 5-FC was reduced to 27.9+/-4.2 micromol.L(-1) in 1000 M.O.I AdCEACD infected Lovo cells and 24.8+/-7.1 micromol.L(-1) in 100 M.O.I AdCMVCD infected Lovo cells, P【0.05, P【0.01, respectively). The CEA-nonproducing Hela cells had no effect after infection with AdCEACD, but Hela cells had the cytotoxic sensitivity to 5-FC after infection with AdCMVCD (The IC(50) of 5-FC in parent Hele cells and Hela cells infected with AdCMVCD at 10 M.O.I was 】15000 and 214.5+/-31.3 micromol.L(-1), P【0.001). AdCEACD/5-FC system also had bystander effect, and the viability was about 30 percent when the proportion of transfected cells was only 10 percent. CONCLUSION: The recombinant adenovirus vector AdCEACD has the character of cell type-specific gene delivery. The AdCEACD/5-FC system may become a new, potent and specific approach for the gene therapy of CEA-positive neoplasms, especially colon carcinoma.
瞄准：到杀死 CEA 积极颜色明确地使用 E 关口 i cytosine 脱氨基酶(CD ) 的表面的癌房间自杀基因，新复制缺乏的 recombinant adenoviral 向量在在哪个 CD，基因在 CEA 倡导者下面被控制被构造并且它的在里面 vitro 细胞毒素的效果被评估。方法：梭原生质标志包含 CD 基因和 CEA 基因的规章的顺序在 293 房间紧张与侵入人体气管粘膜的病菌染色体 DNA 的右手臂被构造并且重新结合。点弄污， PCR 被用来识别积极的匾。侵入人体气管粘膜的病菌的纯化被执行与在 CsCl 步坡度和滴定极端集中与匾形成试金被测量。细胞毒素的效果是有 MTT 方法的 assayed， 5-FC 的百分之五十抑制集中(IC (50 )) 用一个曲线试穿参数被计算。表面的癌房间线，是生产 CEA，和 CEA-nonproducing Hela 房间衬里的人的颜色在 cytological 测试被使用。确定的 recombinant 侵入人体气管粘膜的病菌向量 AdCMVCD， CD 基因在 CMV 倡导者下面在被控制，被用作病毒控制。量的结果被表示为平均数的吝啬的 +/- SD。统计分析用 ANOVA 测试被执行。结果：需要的 recombinant 侵入人体气管粘膜的病菌向量被称为 AdCEACD。点弄污和 PCR 的结果证明 recombinant 侵入人体气管粘膜的病菌包含了 CEA 倡导者和 CD 基因。病毒 titer 是大约 5.0 X 10 (14 ) pfu/L (在纯化以后的 -1) 。 生产CEA Lovo 房间对 5-FC 敏感并且与 AdCEACD 和 AdCMVCD 在感染以后有一样的细胞毒素的效果(在父母 Lovo 房间，感染 100 M.O.I AdCEACD 的 Lovo 房间和感染 10 M.O.I AdCMVCD 的 Lovo 房间的 5-FC 的 IC ( 50 )价值是 >15000 ， 216.5+/-38.1 和 128.8+/-25.4 micromol.L (-1), P<0.001 ，分别地)，并且当侵入人体气管粘膜的病菌的 m.o.i 被提高时， 5-FC 的 cytotoxicity 因此增加了( 5-FC 的 IC ( 50 )的价值被归结为 27.9+/-4.2 micromol.L (在 1000 M.O.I AdCEACD 的-1)感染了 Lovo 房间和 24.8+/-7.1 micromol.L (在 100 M.O.I AdCMVCD 的-1)感染了 Lovo 房间， P<0.05 ， P<0.01 ，分别地)。CEA-nonproducing Hela 房间没与 AdCEACD 在感染以后有效果，但是 Hela 房间与 AdCMVCD 在感染以后有细胞毒素的敏感到 5-FC (在在 10 M.O.I 感染 AdCMVCD 的父母 Hele 房间和 Hela 房间的 5-FC 的 IC (50 ) 是 >15000 和 214.5+/-31.3 micromol.L (-1), P<0.001 ) 。AdCEACD/5-FC 系统也有旁观者效果，并且当 transfected 房间的比例仅仅是 10% 时，生存能力是大约 30% 。结论：recombinant 侵入人体气管粘膜的病菌向量 AdCEACD 有房间的特性类型特定的基因交货。AdCEACD/5-FC 系统可以变得一新，为 CEA 积极的瘤的基因治疗的有势力和特定的途径，特别结肠癌。
作者
Li-Zong Shen Wen-Xi Wu Qiang Ding Yi-Bing Hua,Department of General Surgery,The First Affiliated Hospital of Nanjing Medical University,Nanjing,210029,Jiangsu Province,China De-Hua Xu Zhong-Cheng Zheng Xin-Yuan Liu,Shanghai Institute of Biochemistry and Cell Biology,The Chinese Academy of Sciences,Shanghai,200031,China Kun Yao,Department of Microbiology and Immunology,Nanjing Medical University,Nanjing,210029,Jiangsu Province,China
基金
the Creation Foundation of Nanjing Medical University,No.Cx9905
