【Abstract】ObjectiveSome studies have demonstrated that recombinant adenoviruses are efficient vectors for gene transfer to the venous wall and AdCMV.tk encoded thymidine kinase can be used to reduce restenosis. In this study AdCMV.tk was apply to human vein smooth muscle cells (SMC) and organ cultured saphenous veins to study its effects on proliferation of SMCs and reduction of intimal hyperplasia. MethodsThe adenovirus vector transferred tk gene and mark gene lacZ to the SMC of human saphenous veins and organ cultured vein segments. Various concentrations ganciclovir (GCV) were contained in culture media. The efficiency of gene transfer was studied by using Xgal staining. The proliferation of SMC was monitored by the method of trypan blue exclusion. The bystander effect was observed by mixed cell culture. After vein segments treated by AdCMV.tk+GCV and cultured for 14 days, HE and VG staining were carried out and intimal thickness was analysis by computer image system. ResultsAdenovirus vector could infect saphenous vein SMC efficiently both in cultured SMCs and organ cultured vein segments. Gene expression sustained 14 d at least. The inhibition of SMCs proliferation in vitro was a positive correlation in GCV concentrations and the levels of tk expression. The proliferation of SMCs transfectered lacZ wasn’t restrained by GCV (P＜0.05). In mixed cell experiment there was at least 55% reduction in total cell number when as few as 10% of the cells express tk. Assessment of this “suicide gene strategy” in saphenous vein organ culture model demonstrated that veins treated with AdCMV.tk+GCV had a significant reduction at 14 days in the intimal thickness compared to control group (P＜0.01). ConclusionThe results suggest that adenovirusmediated gene transfer of tk along with GCV administration may be a useful strategy to treat the proliferation of intimal hyperplasia of transplanting saphenous veins. Bystander effects are amplified by AdCMV.tk/GCV gene therapy system.
【摘要】 目的 觀察已構建的含胸苷激酶（TK）自殺基因的重組腺病毒（ADV-TK）對肝癌細胞的體外殺傷作用和對肝癌裸鼠移植瘤的治療效果。 方法 將ADV-TK體外感染人肝癌細胞株SMMC-7721，噻唑藍（MTT）法檢測受感染的SMMC-7721細胞被不同濃度更昔洛韋（GCV）作用后的細胞存活率情況。構建肝癌SMMC-7721裸鼠移植瘤模型，觀察腫瘤注射重組腺病毒ADV-TK結合GCV治療移植瘤的變化。 結果 相同滴度的重組腺病毒與不同濃度的GCV作用于肝癌細胞株SMMC-7721后，MTT法檢測到細胞的存活率隨著GCV濃度的增加而不斷降低。動物實驗中ADV-TK治療組腫瘤體積明顯小于對照組（ADV-null及NS）（Plt;0.01）。 結論 重組腺病毒ADV-TK對肝癌SMMC-7721細胞的體外增殖和裸鼠體內的移植瘤生長均有明顯的抑制作用。【Abstract】 Objective To explore the inhibitory effect of recombinant adenovirus containing TK gene (ADV-TK) on transfected human liver cancer cells SMMC-7721 in vitro and murine transplanted hepatocarcinoma in vivo. Methods SMMC-7721 cells transfected with ADV-TK were exposed to medium with GCV. The cell viability was measured by MTT assays. In the established model of SMMC-7721 human liver cancer, nude mice underwent intratumoral injection with 1 109 pfu ADV-TK, the control vector (ADV-null) or normal saline (NS) and again 7 days later, twice for all. GCV was given at a dose of l00 mg/(kg?d) on the following day of injection for 10 days. The tumor inhibitory effect was observed by measuring the tumor sizes. Results After transfected by ADV-TK in vitro, and combined with GCV, the cell growth of SMMC-7721 cell were significantly suppressed. The result of in vivo assay showed that tumor volumes in treatment group were apparently smaller than that in the control group (Plt;0.01). Conclusion Recombinant adenovirus combined with GCV shows a significant inhibitory effect on SMMC-7721 cells in vitro and murine transplanted hepatocarcinoma in vivo.
Objective To construct replication-defective adenovirus containing tk gene (ADV-tk). Methods Recombinant adenovirus of ADV-tk was constructed using homologous recombination in cells. After the interested tk gene fragment in the recombinant plasmid obtained was confirmed by PCR, the titre of purified recombinant adenovirus was detected. In vitro study, tk gene in SMMC7721 cells transfected by ADV-tk was investigated by RT-PCR. In vivo study, ADV-tk was injected intraperitoneally into BALB/c nude mice with liver cancer and apoptosis cells in tumor were observed. Results Recombinant adenovirus containing ADV-tk was proved successfully. The titre of purified recombinant adenovirus was 1.4×1010 pfu/ml. In vitro study, tk was integrated and expressed by SMMC-7721 cells. In vivo study, with the injection of ADV-tk, apoptosis cells in tumor increased. Conclusion A replication-defective adenovirus containing tk gene is successfully constructed, which may useful for further research on tumor suicide gene therapy with ADV-tk.
Objective To amplificate,clone and sequence the thymidine kinase (TK) gene of herpes simplex virusⅡ(HSVⅡ); to construct and appraise the fusion gene eukaryon expression vector, pcDNA3/HSVⅡ TK/angiostatin. MethodsThe Hep2 cells were infected by HSVⅡ Sav strain. HSVⅡ genomic DNA was purified from the Hep2 cells suspension and used as template to run PCR for TK gene amplification. The amplified products were cloned into PC DNA3 vector and sequenced. The vector pcDNA/HSVⅡ TK was cut by endonuclease. The gained TK gene was cloned into eukaryon expression vector. pcDNA3/angiostation, which had been constructed. ResultsCoding region of HSVⅡTK gene consisted of 1 128 bp except stop code, it encoded 376 amino acids.After cutting the new vector by endonuclease Hind Ⅲ and BamH Ⅰ,we gained the following gene fragment: 1000 bp (TK) and 700 bp (angiostation).Conclusion The fusion gene eukaryon expression vector， pcDNA3/HSVⅡ TK/angiostatin has been constructed.