【Abstract】Objective To study the antitumor activity of HSVtk/CD combinative gene toward human cholangiocarcinoma in vivo. Methods Nude mouse models with transplanted subcutaneous cholangiocarcinoma were constructed and divided into 4 groups randomly, each group had 8 mice. Adenovirus solution free from suicide gene was injected in subcutaneous tumors of each mouse of control group. Adenovirus solution containing cytosine deaminase (CD), thymidine kinase (tk) and HSVtk/CD fusional gene were injected into single suicide gene either HSVtk or CD was transinfected into the tumor cells by injecting viras into subcutaneous tumor of mice of CD gene,tk gene and fused CD and tk gene group respectively. 24 hours after the injection, 5fluorocytosine (5FC) and ganciclovir (GCV) were injected introabdominally in each mouse. Growth of the tumors were monitored.Results Tumor growth of the genetransfection groups was suppressed in different degrees. Compared with the control group, the suppressing rates of the genetransfection groups were 41.2%, 55.7% and 70.0% respectively (P＜0.05). Histological examination showed good tumor growth in the control group, and tumor necrosis in the other 3 groups, particularly obvious in the group transfected with pAd(HSVtk/CD).Conclusion Combinative gene system has a b antitumor effect on cholangiocarcinoma in vivo. But it’s not powerful enough to eliminate tumor thoroughly because of insufficient “Bystander effect”.
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.
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.
【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.
ObjectiveTo preliminarily investigate the expression of thymidine kinase 1 (TK1) in serum of patients with lung cancer and patients with acute exacerbation of chronic obstructive pulmonary disease (COPD), and analyze the relationship of TK1 level with gender, age and smoking.MethodsFifty-two patients with lung cancer and 64 patients with acute exacerbation of COPD, who admitted in this hospital from January 2017 to April 2018 and underwent serum TK1 measurement, were retrospectively collected and analyzed. The serum level of TK1 in the patients was detected with immunoblot-enhanced chemiluminescence method.ResultsThe TK1 expression level had no relationship with gender, age or smoking (P>0.05). A total of 14 patients were tested positive for TK1 (TK1 level>2 pmol/L) in the lung cancer patients and the positive expression rate was 26.9%; a total of 23 patients were tested positive for TK1 in the COPD patients and the positive expression rate was 35.9%. The TK1 expression level had a more obvious increase in the patients with acute exacerbation of COPD than that in the patients with lung cancer, but there was no statistical difference between the two groups (U=1 482, P=0.314).ConclusionsThe TK1 expression level is not affected by patient’s gender, age or smoking. The TK1 expression level in patients with acute exacerbation of COPD is higher than that in patients with lung cancer. Abnormal cell proliferation in patients with acute exacerbation of COPD may be stronger than that in patients with lung cancer, but further studies are needed.