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Experimental Gene Therapy of Keloid in Vitro and in Vivo Using Recombinant Adenovirus Coding for Fas Gene

Author: LuFeng
Tutor: GaoJianHua
School: First Military Medical University
Course: Plastic Surgery
Keywords: Gene therapy Adenoviral vector Bacteria Fas keloid in vitro in vivo
CLC: R622
Type: PhD thesis
Year: 2004
Downloads: 88
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Abstract


Background/Objective:Fibroblasts are testified to be the functional cells which contribute to the pathogenesis of keloid.The unbalance of proliferation and apoptosis in fibroblasts may directly lead to the accumutaion of fibroblasts in keloid tissue. Apoptosis mediatied by Fas has been shown to play an important role in controlling cell proliferation and apoptosis in fibroblasts. More and more researches have been emphasized in the relationship between Fas gene and keliod hi recent year. Our group is the first organization who reported this relationship, the researches indicated that 1.Fibroblasts derived from keloids showed highly expressed Fas antigen and resistance to apoptosis induced by FasMcab in contrast to hypertrophic scars and normal skins. 2. At the same time, low expression of Bcl-2 protein was testified in all samples. 3.Research on the "death signal transduction"mediated by Fas indicated that the Fas signal transduction is blocked upriverly. 4.Mutations in Fas gene were identified in keloid-derived fibroblasts by PCR-SSCP and DNAsequencing which may lead to loss of function in Fas gene, and mutations almostly locate in the "death domain"of Fas gene. 5. we have successfully generated pcDNA-3.1-Fas using molecular cloning technique. And the reconstructed plasmid was transfected into the keloid-derived fibroblasts with the help of lipofectamine. Then the dysfunctional Fas gene was taken place and the blocked Fas signal was reconstructed. But the Fas gene tranffered by lipofectamine can not gain stable expression because of its low transfection rate.In this study, we plan to generate recombinant adenovirus with human Fas gene and transfect the Fas gene into keloid-derived fibroblasts, then Fas gene may obtain stable expression and take place of the dysfunctional protein, reconstruct the blocked Fas signal.Methods:1. Fas gene was cut down from the PcDNA3.1-Fas plasmid, then we constructed recombinant adenovirus vector using PDC 315 system. Recombination was obtained in 293 cells then the recombinant adenovirus Ad-Fas(T) was amplified and purified.2. We used a new bacterial homologous recombination system (Ad-Easy system) to construct recombinant adenovirus vector. Fas gene was cut down from the PMD-T-Fas plasmid and then transferred to track plasmid. Recombination was completed in bacteria BJ5183 and recombinant adenovirus Ad-Fas(B) was produced in 293 cells.3. Construct recombinant adenovirus Ad-Fas(C ) as control which does not carry any functional gene.4. Using MTT method, We evaluate growth status of keloid derived fibroblasts as they were transfected by Ad-Fas(T), Ad-Fas(B) and Ad-Fas(C ).5. With the help of the flow cytometer and Western Blot, changes of expression of Fas gene were detected after Fas gene was transfected into keloid derived fibroblasts by Ad-Fas(T), Ad-Fas(B) and Ad-Fas(C ).6. With the help of the flow cytometer, we detected the apoptosis in keloid derived fibroblasts which was firstly transfected by Ad-Fas(T), Ad-Fas(B) Ad-Fas(C ) and then exposed to FasMcab.7. Reconstruct the keloid model by using athymic nude mice.8. Detect the Changes of tissue transplanted into the athymic nude mice by general observation, pathological research and electronic microscope examination.9. Carry out the gene therapy strategy in vivo including infection of Ad-Fas(T), Ad-Fas(B) and Ad-Fas(C ) followed by other additional measures then general observation, pathological research ,electronic microscope examination was fulfilled as well to uncover the changes of tissue transplanted into the athymic nude mice.Results:1. Fas gene was successflly cut down from the PcDNA3.1-Fas plasmid and constructed into recombinant adenovirus vector using PDC 315 system. Recombination was successfully obtained in 293 cells then the recombinant adenovirus Ad-Fas(T) was amplified and purified.2. Fas gene was successfully cut down from the PMD-T-Fas plasmid and then transferred to track plasmid using bacterial homologous recombination system (Ad-Easy syste

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CLC: > Medicine, health > Surgery > Plastic Surgery (repair surgery ) > Plastic surgery school
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