Effect Of LV-mIL12Transfection Of Rat Pedicled Siea Flap By Intra-artery Perfusion On SHZ-88Breast Cancer Cell Growth In Vivo

Gene therapy play an important role in antitumor treatment, and the targeted gene therapy become a focus of gene therapy research because of its targeted effect, very low systemic reaction. Flap is a common tool for plastic surgeons, which has important effects in reconstruction of soft tissue after chronic wounds and tumor excision. The flap was furthered by gene modify as the reconstructional tool and the therapeutic tool. In transplant medicine and interventional medicine, it has been reported that transfect of topical tissue by perfusion of target gene can promote the expression of therapeutic proteins, but the literature is less in studies on plastic surgery. In our study, on the basis of modified rat pedicle superficial inferior epigastric artery (SIEA) flap, we investigate the feasibility and safety of transfect of lentivirus medicated exogenous gene (lentivirus mediated enhanced green fluorescent protein, LV-EGFP) by intra-artery perfusion, and evaluate the transfect conditions. We further constructed recombinant lentivirus medicated lnterleukin-12plasmid (LV-mIL12) and transfected it in SIEA flap by intra-artery, and observe its antitumor effects. The study is composed of the following four aspects:1. Construction of intra-artery perfusion model of rat pedicle SIEA flapBased on the former research of the blood supply system of rat pedicle SIEA flap, we confirmed the blood supply system by methylene blue perfusion and the operational procedures were designed to produce the closed circuit transiently isolated with general circulation in rat pedicle SIEA flap, and the effects on survival of pedicles were observed by perfusing phosphate-buffered saline(PBS) through saphenous artery and saphena. It was indicated that, the occlusion time of flap were designed to120min, and the pedicle SIEA flap was healed normally after perfusing PBS,, and no significant difference was observed in neutrophil counts between different groups undermicroscope (P>0.05).2. Lentivirus medicated exogenous gene EGFP (LV-EGFP)transfected rat pedicle SIEA flap by intra-artery perfusionRat and human primary cell lines representative of a composite tissue flap cellular architecture were transduced using LV-EGFP in vitro:rat aortic endothelial cell, RAEC,rat dermal fibroblast, RDF,human dermal fibroblast, HDF,human umbilical vein endothelial cell, HUVEC. The transduction efficiency and gene expression level in different TOI and MOI were observed and optimized. The results showed that the transduction efficiency of HDF, HUVEC can be more than50%when MOI=50, more than80%when MOI=200. For RDF and RAEC, the transduction efficiency can be70%when MOI=200. We concluded that all of the four cells can be transfected effectively when MOI=200with a good cell vitality. HDF and HUVEC transduced after2hours can achieve a satisfactory transduction efficiency when MOI=200. The transduction efficiency of RDF and RAEC became better as the longer transduction time (within12h), and can be above50%when transduced after2h.The study of LV-EGFP transduced rats pedicle SIEA flap by the artery perfusion:the test rats were divided randomly into4groups based on the applied perfusion solutions: LV-EGFP (1.0×108TU/ml,5.0×108TU/ml,1.0×109TU/ml), PBS. The flap was transduced for120min. The evaluation of exogenous genes expression and distribution in flap tissue was performed on7,14,21,28,40days after perfusion. The results show that no EGFP expression was found in1.0×108TU/ml group and PBS group at any time point, there was a weak EGFP expression in5.0×108TU/ml (C group)14days after perfusion, but disappeared in21days. There was EGFP expression in1.0×109TU/ml (D group) through7to40days. RT-PCR results show EGFP mRNA was existed in flap and the surrounding tissues of flap, not found in liver and kidney.3. Construction and identification of LV mediated mIL12and assay biological activity of mIL12.Selecting mlL12as the purpose gene, pORF-mIL-12as template, we used PCR to obtain purpose gene, which recombined with LV vector. Recombined lentiviral vectors mediated mIL12was constructed with a three-plasmid lentivirus packaging system. HDF, RDF, HUVEC, RAEC was transduced with LV-mIL12, the mIL12expression was detected by Western Blot, ELISA. The levels of INF-γ were determined in supernatants of transduced cells to assess the biological activity of mIL12. The results showed that recombined lentiviral vectors mediated mlL12was constructed successfully with a three-plasmid lentivirus packaging system, mIL-12cDNA can express in cell. The virus titer was2.00E+8TU/ml. LV-mIL-12can transduced effectively HDF, RDF, HUVEC, RAEC which can secret mIL-12with biological activity.4. Effect of LV-mIL12transfection of rat pedicled SIEA flap by intra-artery perfusion on SHZ-88breast cancer cell growth in vivoTo establish the rat breast cancer model of SHZ-88by subcutaneous injection. The rats were divided randomly into three groups based on the applied perfusion solutions: LV-mIL12, LV-EGFP, PBS. The SIEA island flap was perfused with the different perfusion solutions according to the previously described procedure. Tumor growth and survival of the SIEA flap was observed on days1-40. The mlL-12expression in tissue and surrounding tissue was assessed by ELISA, IFN-λ levels in tumor tissue and tumor volum were determined to evaluate tumor response to treatment. The results show:mIL12is expressed in transduced flaps and surrounding tissue, mIL12expression flap> surrounding tissue> control. mIL12transduced flaps suppressed the growth of tumors at the early time(7-15days).In general, the study establishes a simple and stable intra-artery perfusion model of rat pedicle SIEA flap. Lentivirus vector can transfect exogenous gene EGFP to rat pedicle SIEA flap by intra-artery perfusion. Recombined lentiviral vectors mediated mIL12was constructed successfully with a three-plasmid lentivirus packaging system, mIL-12cDNA can express in plasmid. LV-mIL-12can transduced effectively HDF, RDF, HUVEC, RAEC which can secret mlL-12with biological activity. mIL12is expressed in transduced flaps and surrounding tissue. mIL12transduced flaps suppressed the growth of tumors at the early time(7～15days).