| BackgroundSuicide gene therapy is one of the most promising methods of cancer gene therapy which transfers suicide genes, namely prodrug-activating enzyme genes that are found only in viruses and bacteria but not in mammalian cells, into cancer cells by some gene engineering methods to express some kinds of enzymes which could catalyze nontoxic prodrugs into cytotoxic substance so as to kill the cancer cells. It has been found that not only suicide gene-transfected cancer cells but also non-transfected cells will be damaged during the suicide gene therapy by means of the direct killing effect and so-called bystander effect, so the anti-tumor effect is pretty wonderful with mild side-effects.Herpes simplex virus thymidine kinase (HSV-TK)/ ganciclovir (GCV) and E. coli cytosine deaminase (CD)/5-fluorocytosine (5-FC) are the two most mature suicide gene therapies so far and several clinical test protocols have been approved by FDA in U.S.A.. With regards to the fact that different types of tumor cells have different sensitivity to different suicide gene therapies and drug resistance is easy to occur at the presence of a single suicide gene therapy system, combined use of different suicide gene systems is recommended and CD/TK double suicide gene is most interesting. There are several advantages of using CD/5-FC and TK/GCV at the same time, such as achieving synergetic effect, improving anti-tumor effect and avoiding drug resistance, etc.The specific expression of suicide genes in the tumor cells so as to selectively damage tumor cells could be realized by taking advantage of some tumor specific transcription modulating elements, such as promoter and enhancer. For example, AFP promoter was commonly applied in the suicide gene therapy for hepatic cancer and erb2 promoter for breast cancer. However, most of the common promoters used nowadays are specific for some kind of cancer and their usage are relatively limited. It has been revealed that vascular endothelial growth factor (VEGF) over-expresses in almost all thesolid cancer cells but not in normal cells and many research results have demonstrated that VEGF promoter could make genes of interest over-express exclusively in tumor cells.Most of the vectors for suicide gene therapy are viral vectors. At present, adenoviruses, adeno-associated viruses, herpes simplex viruses and retroviruses are frequently used. Owing to some special biological characteristics, such as high viral titer, large capacity, being able to infect both dividing and non-dividing tumor cells, not integrating into the host genome and being applied in vivo experiment, etc., adenoviruses are widely used in every kind of gene therapy, especially in the suicide gene therapy. Before the homologous recombination in bacteria was found, recombinant adenoviruses could be constructed only in a few large research centers. With the development of molecular biology and gene engineering, constructing recombinant adenoviruses have become much simpler, especially after AdEasier-1 system was used.So in my experiment, a new kind of recombinant adenovirus containing CD/TK fusion suicide gene under the control of VEGFP is going to be constructed by a simplified and high performance AdEasier-1 system which is kindly given by professor Belt Vogelstein and the in vitro anti-tumor effect of the recombinant adenovirus containing CD/TK double suicide gene driven by VEGFP on LOVO tumor cells would be examined too.Objectives1. To construct recombinant adenoviral plasmids containing CD/TK fusion suicide gene under the control of VEGFP by a simplified and high performance AdEasier-1 system, which were then packaged and amplified in a mammalian packaging cell line 293 cells. Finally, the recombinant adenoviruses were purified by CsCl banding procedure and proved to be correct by PCR.2. Clone forming test and MTT method were used to examine the in vitro anti-tumor effect of the recombinant adenovirus containing CD/TK double suicide gene driven by VEGFP on LOVO tumor cells.Methods1. Construction of...
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