Adenovirus-mediated Gene Transfer Of Human Interleukin-10 Combined With Interferon-γ Inhibits Proliferation Of Vascular Smooth Muscle Cells And Its Mechanism

The therapeutic effectiveness of vascular intimal hyperplasia is by no means good when only considering single factor, combined treatment concerning multiple factors is one of the trends in development. Both interleukin-10(IL-10) and interferon-γ(IFN-γ)can inhibit vascular smooth muscle cell(VSMC) proliferation under particular conditions. IL-10 can inhibit the in vivo expression of IFN-y, then enervate its function in inhibiting VSMC proliferation. So, we consider the complement of IFN-y when using IL-10 to treat vascular intimal hyperplasia. The half life of merchandizing recombinant IL-10 protein is short, but the administration of the replication deficient adenovirus-IL-10 construct results in an increase in serum or local IL-10 concentrations, overcoming the constraint imposed by the short half life of exogenous IL-10. Our study includes three parts: (1) to get interest gene by cloning human IL-10 cDNA from in-vitro cultured peripheral blood mononuclear cells, for the preparation of construction of recombinant adenovirus vector; (2) the construction of recombinant adenovirus vector expressing both human recombinant green fluorescent protein and human interleukin-10; (3)Transfect in-vitro cultured human umbilical artery smooth muscle cells induced by epidermal growth factor, simultaneously adding IFN-γ of 200~u/ml end concentration to the culture medium, to investigate the effect of IFN-γ combined with adenovirus-mediated gene transfer of human IL-10 on the proliferation of vascular smooth muscle cells, and study the relevant mechanism, to explore ideal combined gene therapy methods to inhibit neointimal formation, for the improvement of treatment effect of vascular intimal hyperplasia.Part oneThe Cloning of human interleukin-10 cDNA from in vitro-cultured peripheralblood mononuclear cellsObjective: To clone human interleukin-10 cDNA from in-vitro cultured peripheralblood mononuclear cells for subcloning and gene therapy study. Methods:peripheral blood mononuclear cells were separated from human peripheral venousblood with lymphocyte separation fluid and in vitro cultured for 24 hours with RPMI1640 containing concanavalin A, RNA was extracted from PBMCs, human interleukin-10 cDNA was amplified with RT-PCR and cloned into pUCm - T vector, recombinant were transformed into competent cells DH5a. Prepare miniprep DNA after blue - white color screening. PCR amplifying > restriction mappings recombinant sequencing were performed. Results: The 560bp fragment amplified with RT - PCR is the same as the anticipated one in size, miniprep DNA was prepared from randomly isolated white colony , the fragment got from PCR amplification and restriction mapping is the same as the anticipated one in size, sequencing shows its complete homology to hIL-lOcDNA published in Genebank . Conclusion: The cloning of human interleukin-10 cDNA from PBMCs and recombination with pUCm - T vector is feasible and relatively simple , provides dependable prerequisite for further subcloning and gene expression study .Part twoThe construction of recombinant adenovirus expressing human interleukin-10 and GFP by homologous recombination in BacteriaObjective: To produce recombinant adenovirus expressing human recombinant green fluorescent protein and human interleukin 10 by homologous recombination in bacteria. Methods: hIL-lOcDNA was amplified from pUCm-T/hlL-lOcDNA using polymerase chain reaction, and cloned into shuttle plasmid pShuttle-IRES-hrGFP-1, kanamycin resistancy screening for positive recombinant plasmids, which were linealized with restriction enzyme Pme I, and transformed into competent bacteria BJ5183-AD-1 containing pAdeasy by electroporation after determining that the insert's sequence is correct by Not I and Xhol I restriction enzyme digestion sequencing and BLAST; prepared recombinant adenovirus plasmids were transformed into ultra-competent XLIO-Gold cells, amplified positive recombinant adenovirus plasmids were transfected to AD-293 cells for packaging after being linearized with restriction enzyme Pac I. PCR was used to determine the target gene. The titer of the recombinant Ad was measured by expression forming units' method, IL-10 concentration in transfected VSMC supernate was measured by enzyme linked immunosorbent assay(ELISA). Results: The recombinant shuttle plasmids contained interest gene of hIL-10, recombinant Ad had 30kb and 3kb fragments after digestion with restriction enzyme Pac I, PCR indicated that the recombinant Ad...