| OBJECTIVE: Wound healing is of major important to the survival and clinical outcome of burn patients, but it meets with many problems during treatment. In order to probe into the effect of gene therapy for treatment of wound healing, combined exogenous gene of IGF-I and HSV-tk transported by liposome were conducted to the wound of scald rats. The gene expression of IGF-I and HSV-tk were detected respectively. In the meantime, the benefit of IGF-I gene transduction on wound healing, and inhibition of tk gene transfer on fibroblast were observed. The gene therapy of this study has made a positive trial in burn and plastic surgery. METHODS: The study is composed of two parts.The first part tends to actualize construction of recombinant eukaryotic expression vector of IGF-1 and HSV-tk. In this period, IGF-I and tk gene from prokaryotic plasmids were amplified with PCR techniques, and were cloned to eukaryotic plasmid of pcDNA3.l respectively, then eukaryotic recombinant plasmid of pcDNA3.1/IGF-I and pcDNA3.1/tk were constructed successfully.There were 5 steps in the first part. First, Harvest for DNA fragments of IGF-I and HSV-tk from prokaryotic vectors of pUChIGF-I and pUChHyTk by PCR methods respectively. Second, Recombination of DNA fragments of IGF-I and HSV-tk with plasmid pcDNA3.1 in vitro. Third, Transformation of permissive cell of Bacillus coli conducted byrecombinant pcDNA3.1/IGF-I and pcDNA3.1/tk. Fourth, screening and evaluation of transformant. Finally, Assay and analysis of gene sequence of IGF-I and tk in recombinant plasmid pcDNA3.1/IGF-I and pcDNA3.1/tk.The second part aims to probe into the effect of combined gene transfer of IGF-I and tk conducted by liposome on wound healing of scald rat.In this part, pcDNA3.1/IGF-I and pcDNA3.1/tk construct transported by liposome were injected subdermally in wound region of scald rats. Changes of body weight, tendency of wound healing were observed carefully within 5 weeks postburn. The expression of IGF-1 gene transfer was observed with immunohistochemical staining, and expression of tk gene transfer was detected by RT-PCR technique; on the other hand, after GCV was injected, apoptosis of fibroblast positive to tk gene transfer was observed under transmission electron microscope.Results: The results of agarose gel electrophoresise evidenced that IGF-I and tk gene fragment was correctly inserted into the cloning site of the eukaryotic expression plasmid vector pcDNA3.1 respectively. The gene fragments from pcDNA3.1/IGF-I and pcDNA3.1/tk, digested with Hind III and EcoR I restriction enzymes, were as same as those digested with the same restriction enzymes from pUChIGF-I and pUChHyTk. Furthermore, DNA sequence analysis of IGF-I and tk gene indicated that target gene was corrected reconstructed separately.The results of animal experiment showed that IGF-I gene transported by liposome expressed positively in fibroblast in the burn wound withimmunohistochemical staining, and expression of tk gene transfer can also be detected by RT-PCR technique; meanwhile, after GCV was injected, apoptosis of fibroblast positive to tk gene transfer was observed under transmission electron microscope.There were no significant differences in the serum IGF-I concentrations among treatment groups and control group (p>0.05). The speed of wound healing for animals injected with IGF-I reconstruct was faster than those which didn't injected IGF-I (p<0.05); The body weight of the animals in groups injected with IGF-I (group A,C1,C2) hadn't a loss in 5 weeks postburn, but those which didn't injected IGF-I (group B,D) had a loss in body during post burn days (p<0.05); After GCV injection, apoptosis of fibroblast positive to tk gene transfer could be detected under transmission electron microscope. CONCLUSION: Eukaryotic recombinant plasmid of pcDNA3.1/ IGF-I and pcDNA3.1/tk were constructed successfully by subcloning method. The recombinant DNA plasmids were introduced into burn wound of rat respectively or collectively by liposome-mediated transfection. Positive expression...
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