To Promote The Revascularization Of Random Skin Flap By Gene Therapy

Gene therapy is a promising method to treat disease, which has been developed since the last decade of the twenty-century. In contrast to the traditional therapy with protein drugs, it works by transferring the exogenous heritable material such as DNA or RNA into cells, which would upregulate or downregulate the target protein expression or pathway, therefore treat the disease. Researches of gene therapy are taken in almost every medical field at present, especially in the treatment of such disease, as heredity disease, tumor, cardiovascular disease, and the results are very exciting. However, researches in the field of tissue transplanting and trauma healing are still at the beginning, so we designed the current study of promoting the revascularization of random pattern of skin flap by transfecting plasmid encoding VEGF165. VEGF165 is well known for its ability to stimulate the development of new blood vessels. The following experiments were done to acquire the information about the constructed vector, the dose and routine of administration of the gene drug. Our results will help to establish an optimal protocol that can be easily utilized in tissue transplantation or similar situations related to angiogenesis.VEGF165 cDNA was amplified by PCR (polymerase chain reaction) from the human fetal liver cDNA libraries. Product of the PCR reaction was analyzed by 1.0% agarose gel electrophoresis, showing the expected band of 590 bp. The target fragment was isolated and purified from the gel and cloned into a pUC19 vector. The recombined plasmid was purified and sequenced. The fragment with correct DNA sequence was selected, and then cloned it into a mammalian expression vector pcDNA3.1/Zeo(+). Preparation and purification of pcDNA3.1/Zeo(+)-VEGF165 was operated under the endofree plasmid Mega protocol of QIAGEN plasmid purification handbook. Transient transfection of the established mammalian expression system pcDNA3.1/Zeo(+)-VEGF165 was mediated with lipofectamine 2000, using the human embryonic kidney cell line 293 cultured in medium DMEM. After transfection of 24h, 48h, 72h, the cell lysates and the culture media were collected respectively, and were subjected to SDS-PAGE gel for AgNO₃ staining and western blotting to assay the expression of VEGF gene in vitro. Ten Sprague-Dawley rats was used as the experimental animal to assay the expression of pcDNA3.1/Zeo(+)-VEGF165 in vivo. 5μg recombined plasmid diluted into 100μl physiological salt solution and 20μl Lipofectamine 2000 also diluted to 100μl with physiological salt solution was mixed thoroughly and incubated at room temperature for 20 minutes, then the complexes was injected into the subcutaneous tissues on the back of every one rat. A series of skin, liver and kidney samples of the rat transfected with...