The Preclinical Study Of Imaging And Suicide Gene Therapy Of HSV1-tk Reporter Gene To Adenocarcinoma Of Lung

OBJECTIVEThe current incidence and mortality of lung cancer has grown rapidly, It has become one reason of the major human deaths, the most prominent of which is the Increase of lung adenocarcinoma. the overall effect of Chemotherapy and drug treatment is less optimistic. The cancer is prone to form local recurrence and distant metastasis,. Gene therapy for lung cancer is an important issue for treatment of lung cancer, in which suicide gene (suicidegene) therapy is considered the most promising method of gene therapy.At present, herpes simplex virus (HSV1-tk) genetic research is the most of many therapeutic genes and the most promising of radioactive nuclides with catalytic "suicide gene". In addition, HSVl-tk has reporter gene features. Thus establishing the nude mice animal model of lung cancer of carrying tk gene, then expressing thymidine kinase TK in vivo, intaked in a amount of exogenous of radionuclides such as 18F, 124I,131I, etc. substrate nucleoside analogues labeled. We can monitor with real-time monitoring the expression of tk suicide gene in target cells by PET imaging which is basis for further in-depth study for the lung cancer suicide gene therapy research. Also, We can for the next double reporter gene imaging of double tracking and monitoring of bone marrow mesenchymal stem cells in vivo feasibility of laying foundation repairCONTENTThe exogenous suicide gene is transduced into human tumor cells in different places and the stable,efficient expression successfully in target cells are the keys of gene therapy for cancer. In addition, the real-time detection of the distribution of suicide gene in vivo with patients and treatment status to the entire treatment process is an integral part. In this study, we transfect HSV1-tk suicide gene and reporter gene into lung adenocarcinoma A549 cells with retroviral vector-mediated,based on previous study.By screening positive transduced cells, re-use dilution to establish the monoclonal lung adenocarcinoma cell line A549-tk which can stable express HSV1-tk gene, Observion the difference of the morphology and growth status between the normal A549 cells and A549-tk cells,. Established lung cancer model of nude mice with carrying tk gene, testing whether the TK gene can express long-term stability in vivoMETHODSHSV1-tk gene of fragment with restriction site of Bgl II, Sall is amplified by Molecular cloning. The retroviral vector pDON-AI-2-Neo which HSV1-tk gene is inserted into, and the plasmid pGP expressing gag-pol and the plasmid pE-ampho expressing env are transfected 293T packaging cells by liposome to form artificial recombinant virus particles carrying pDON-AI-2-Neo-HSV1-tk reporter gene. Then it is transfected A549 lung cancer cells.The transfected cells by G418 selecting positive expression, then with limiting dilution Monoclonal is formed stable expression strain.RT-PCR testing confirmed the expression of exogenous HSV1-tk gene in A549-tk cells is stability in the mRNA level. The morphology observation and cell growth curve are finished with the long passage of cell culture growth.. Cloned tumor cell lines are implanted into nude mice, studying the difference between the normal A549 and A549-tk tumor tumor growth, RT-PCR detects HSV1-tk gene expression of A549-tk tumorRESULTSThe HSV1-tk gene of plasmid pDON-AI-2-Neo-HSVl-tk is consistent with HSV1-tk gene sequences (gi:59974) published by GenBank gene pool with digesting and sequencing.It is usd to merged the plasmid pGP expressing gag-pol and the plasmid pE-ampho expressing envl which were prepared false infectious particles by packaging celPositive selection by the antibiotic G418 and limiting dilution method cultivate a monoclonal cell lines expressing HSV1-tk gene, and RT-PCR confirmed HSV1-tk gene is transfected into A549 cells successfullyWe found that the same shape of two cells are basically, and no significant difference in growth characteristics (P> 0.05) by compareing morphology and the cell growth curve between A549-tk cell line and normal A549 growthA549-tk tumor growth was 100%, that is no different with A549 cells on tumorigenicity.We found that the tumor size and tumor growth between the normal A549 and A549-tk tumors was no significant difference (P> 0.05) by compareing the growth characteristics of two cell tumors. After three passages, RT-PCR analysis showed that the successful establishment of stable and efficient expression of HSV1-tk gene in an animal modelCONCLUTION1 The plasmid pDON-AI-2-Neo-HSVl-tk can be translated and expressed correctly in adenocarcinoma of lung A549 cell.2 HSV1-tk gene in A549-tk cells can be stability expressed after long-term successfully.3 the exogenous gene HSV1-tk imported did not affect the normal physiological function and morphology of A549 cells.4 After exogenous HSV1-tk reporter gene into A549 cells, there is no significant effect for the state on tumor growth.5 The experiment is successfully established an animal mode expressing HSV1-tk gene stablely and efficiently.