双自杀基因重组腺病毒对胰腺癌细胞特异性杀伤作用

Adenovirus mediated fusion gene system driven by KDR promoter kills selectively pancreatic cancer cells

目的 研究腺病毒介导的KDR启动子驱动CD／TK融合基因系统（AdKDRCDTK）对胰腺癌细胞Capan-2特异性的杀伤作用。方法 重组腺病毒体外感染表达KDR的Capan-2细胞株，用不表达KDR的肝癌细胞HepG2做对照，观察其感染效率并以RT-PCR方法检测转基因细胞CDTK的表达，然后给予不同浓度的前药更昔洛韦（ganciclovir，GCV）和5-氟胞嘧啶（5-fluorocytosine，5-FC），MTT法观察该体系对Capan-2和HepG2细胞生长增殖的影响及其旁观者效应；电镜观察细胞的病变；流式细胞仪检测细胞周期的变化和DNA含量的变化。建立Capatv-2裸鼠皮下移植瘤模型，瘤内注射Ad-KDR-CD／TK，腹腔注射前药GCV（50mg·kg^-1·d^-1）和5-FC（500mg·kg^-1·d^-1）14d，观察肿瘤生长抑制效应。结果 腺病毒对两种细胞株的感染率相似，其感染率随腺病毒滴度的增高而递增。RT-PCR方法检测发现转染Ad-KDR-CDTK的Capan-2细胞有目的基因表达。MTT法检测显示前药呈剂量依赖性抑制Capan-2生长，而不表达KDR的肝癌细胞HepG2对前药不敏感，且观察到该体系对Capan-2明显的旁观者效应。电镜下可见Capan-2有凋亡改变。用流式细胞仪测定用药组出现典型的凋亡峰；细胞周期分析显示治疗后细胞G0-G1，期比率增多，G2-M及S期细胞减少。在Capan-2裸鼠移植瘤模型中，该双自杀基因系统能够显著抑制肿瘤的生长。结论 KDR启动子可调控双自杀基因体系选择性杀伤胰腺癌细胞Capan-2，诱导胰腺癌细胞凋亡，并可显著抑制人胰腺癌裸鼠移植瘤的生长。

Objective To evaluate the selectively killing effect of adenovirus fAd） mediated double suicide gene driven by KDR promoter on pancreatic cancer cell Capan-2. Methods KDR-ex- pressing Capan-2 and non-KDR-expressing HepG2 were infected by Ad-KDR-CDTK. The infection rate was observed and the expression of CDTK was detected by RT-PCR. Followed by treatment with 5-FC and GCV,the killing effects were evaluated and bystander effects were analyzed by MTT. The pathological character of cells was observed with electron microscopy and distribution of cell cycle was detected by flow cytometry. An animal model of human pancreatic cancer in nude mice was reproduced. Ad-KDR-CD/TK was injected directly into the tumor. Then,the nude mice were given intraperitoneal injection of GCV （50 mg·kg^-1·d^-1） and 5-FC （500 mg·kg^-1· d^-1） daily for 34 d. The inhibition rates for tumor growth were also calculated. Results The infection rate of the resultant recombinant Ad to Capan-2 and HepG2 cells was not apparently different,and it increased gradually with the addition of multiple of infection （MOI） of Ads. RT PCR demonstrated that there existed the product of CDTK gene in Capan-2 cell infected by Ad-KDR-CDTK. Prodrug could inhibit proliferation of Capan-2 and the effect was dose-dependent,while the infected HepG2 cells were not. There was considerable bystander effect by MTT. Apoptotic peak was also shown by flow cytometry. Morphologic features of apoptosis in Capan-2 cells were displayed via electron microscopy. The analysis of cell cycle revealed that the rate of cells at the G0-G1 phase was increased and the rate at the G2-M and S phase was decreased by treatment with pro-drug. Furthermore,the tumors decreased in size during treatment of double suicide gene system by establish subcutaneous transplanted model. Conclusion The CDTK fusion gene system controlled by KDR promoter have selectively killing effects on the KDR expressing Capan-2 cells and inducing the cell apoptosis. It can significantly inhibit the growth of xenografted pancreatic cancer in nude mice.