目的

探讨CEA启动子(CEA promoter,Cp)在肿瘤细胞特异性启动双自杀基因胞嘧啶脱氨酶(cytosine deaminase,CD)与胸苷激酶(thymidine kinase,TK)的作用。

方法

培养Lovo与Hela细胞，传代冻存。10 μl带有Cp.CD.TK重组腺病毒(recombinant adenovirus with Cp.CD.TK, RA-Cp.CD.TK)病毒液转染Lovo与Hela，荧光显微镜观察，收集细胞提取总RNA, PCR扩增Cp、CD与TK，电泳鉴定。病毒转染Lovo与Hela传代接种96孔培养板，每株细胞分两组，每组24孔：第一组给予5-氟胞嘧啶（5-fluorocytosine，5-Fc）10 mg/L培养液，第二组给予更昔洛韦(ganciclovir,GCV)5 mg/L培养液，光镜观察细胞形态，台盼蓝染色检测细胞活力。

结果

转染病毒的Lovo细胞及Hela细胞均可见绿色荧光，PCR均可扩增出Cp、CD和TK。5-Fc和GCV对Lovo细胞具有很强的杀伤作用，对Hela细胞则微弱，5-Fc实验组Lovo细胞活力平均为8.54%±1.34%，Hela细胞平均为95.05%±2.18%(P＜0.01)。GCV实验组Lovo细胞活力平均为8.38%±1.22%，Hela细胞平均为95.39%±1.64%(P＜0.01)。

结论

RA-Cp.C.T靶向性杀伤CEA⁺的Lovo结肠癌细胞，而对CEA^-的Hela子宫颈癌细胞生长无影响。

Objective

To investigate the CEA promoter’s role on CD and TK for gene treatment of tumors.

Methods

Lovo and Hela cells were cultured, passed, stored in liquid nitrogen, transfected with 10 μl recombinant adenovirus with Cp.CD.TK(RA-Cp.C.T),observed the green fluorescence. The total DNA of transfected Lovo and Hela were extracted and employed as PCR templates for amplifing Cp,CD and TK.The three interest genes were identified with agarose electrophoresis.The Lovo and Hela cell transfected with RA-Cp.C.T were overlayed on 96 well cell culture board, and every strain of cell was divided into two groups (24 wells per group). The first group was cultured with growth medium containing 10 mg/L 5-Fc, and the second group with growth medium containing 5 mg/L GCV. Cell morphologic observation with light microscope and cell vitality measure with trypan blue dying were carried out.

Results

All the Lovo and Hela cells,being transfected by RA-Cp.C.T，presented green fluorescence and had Cp,CD and TK DNA identified by PCR.Both 5-Fc and GCV could kill Lovo cells,but almost no influence on Hela cells.In 5-Fc group,the average vitality of Lovo cell was 8.54%±1.34%，while that of Hela cell was 95.05%±2.18%(P＜0.01). In GCV group, the average vitality of Lovo cell was 8.38%±1.22%，while that of Hela cell was 95.39%±1.64%(P＜0.01).

Conclusion

The suicide gene of CD and TK promoted by Cp kill violently CEA positive Lovo cell, and can’t kill CEA negative Hela cell.