Study On The Targeted Therapy Of Tumour By Retroviral Vector-mediated Thymidine Kinase Gene

With the development of modern industry and the serious pollution of environment, the incidence and death rate of lung cancer has been increasing in the world since 1970s. Lung cancer is the most common malignancy and the first leading cause of cancer-related deaths among people in the world. About 150~180 thousand people die with lung cancer every year. At present, more men can be effectively treated by surgery or radiation. However, The cure rate of lung cancer is still less than 15 percent. In recent years, Whereas significant progress has been made in defining the molecular mechanisms of lung cancer development, the specific molecular pathways involved in lung cancer have not been fully characterized. However, tumor-targeting therapy may lead to effective treatments for lung cancer.One new approach involves gene therapy is using a retroviral vector that carries a suicide gene, such as the TK gene. Retroviruses can stably integrate their genes into proliferating cells instead of quiescent cells. The TK gene product has killing effect only on proliferating cells in which TK gene can efficiently phosphorylate nucleoside analogues and the phosphorylate products act as a chain terminator of DNA synthesis, then leading to cell death. If the HSVTK gene is driven by the tumor-specific promoter, it can cause selective inhibition of tumor cells.The human telomerase catalytic subunit (hTERT) is highly activated in 80~90% of cancer cells, but seldom expressed in normal cells, and it is considered as a broad-spectrum tumor marker. Tumor development requires oxygen and nutrients,which are supplied through neovascularization. The vascular endothelial growth factor (VEGF) plays an important role in tumor development and progression, supplying oxygen and nutrients through neovascularization. It has become a promising molecular target for lung cancer therypy. Herpes simplex virus thymidine kinase (TK) gene is a very important suicide gene with obvious bystander effect. Specific primers were designed and all DNA fragments were obtained respectively. The products were linked into vector pMD18-T vector ,then cloned ,sequenced and analyzed. The results showed that we obtained the purpose DNA fregments.According to Restriction enzymes of the CMVp of green fluorescent protein expression vector PECFP-C1, we designed specific primers, containing Asel & Nhel rstriction sites. A specific DNA fragment about 723bp was obtained. The DNA fragment was linked into vector pMD18-T vector and then transformed into competent host strain Escherichia coli DH5 a . Positive recombinant plasmid was sequenced, and the result showed the complex regulation sequence was correct. The complex regulation sequence, digested from the recombinant pMD-[VEGF]hTERT by Asel & Nhel, was inserted into the CMVp of expression vector PECFP-C1. Then the constructed plasmids were transfected into the human lung cancer cells A549 and normal human liver cells. The ECFP expression was observed only in lung cell lineAccording to restriction enzymes of retroviral vector pLNC-LacZ , the sequence of [VEGF]hTERTTK or hTERTTK , digested from the vector pBluescriptllSK-fVEGFlhTERT-TK with restriction enzymes, was inserted into the retroviral vector pLNC-LacZ, then transformed into competent host strain Escherichia coli DH5 a . Plasmids were isolated from recombinant clones by alkaline lysis, and PCR identification, restriction analysis. The recombinant retroviral vector were transfected into PT67 packaging cells by liposometransfection. The cells were selected in the medium with 500ng/ml G418 after 2 weeks, G418-resistant colonies were obtained and amplified. The supernatant of cell culture was harvested and was used to infect NIH3T3 cells to determin the viral titer of recombinant retrovirus. Results showed that the average titer of viral supernatant was 8.2 X 104CFU/ml and 7.8 X 104CFU/ml .Lung cancer cell line A549 was incubated for 4h in 3ml viral supernatant. The cells were then cultured in a complete medium containing G418(700ng/ml) for one month.The G418-selected pools of cells transfected with virus were plated in 6-well. The cells were treated with 3ml fresh medium in the absence or presence of varying concentration of GCV. The cells were incubated under hypoxia(l%02) for 24h every 2 days. GCV-mediated cell growth inhibition was determined by direct cell number counting and MTT assay. The experimental results revealed that the cell number of group treated with GCV was lower than the group untreated with GCV. And the OD value of group under hypoxia was lower than the group in normoxia. Lung cancer cells were determinated remarkable apoptotic characteristics such as nuclear shrinkage by light microscopy with Giemsa staining, and cells' shape changed obviously. DNA laddering assay, FCM combined with PI and AnnexinV -FITC double pigmention methods were used to observe the inductive effect of lung cancer cells infected by the system of TK gene combined with ganciclovir(GCV). The results showed the DNA ladder demonstrated that the system of TK gene combined with GCV could induce apoptosis in A549 cancer cells. The apoptosis percent of group under hypoxia were higher than another group in normoxia with GCV administration, the cell cycle were exchanged also detected by FACS, but few apoptotic characteristics were observed in control groups without GCV administration.In this study, the results suggests that retrovirus mediated system of TK genecombined with GCV can induce apoptosis and inhibit multiplication of A549 lung cancer cells.