高表达ang1基因的间充质干细胞株的建立及其肺内转化研究

高表达 Ang1基因的间充质干细胞 的建立及其肺内转化研究 徐金富 瞿介明 李惠萍等 同济大学附属上海市肺科医院 复旦大学附属华东医院 High promise for angiopoietin- 1(Ang1) in lung injury intervention n Ang1 decreased in LPS induced lung injury n anti-inflammation: inhibits endothelial permeability, neutrophil adherence and IL-8 production n Anti-apoptosis role to endothelial cells Thurston G, Rudge JS, Ioffe E, et al. Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med. 2000, 6: 460- 463. Daly C, Wong V, Burova E, et al. Angiopoietin-1 modulates endothelial cell function and gene expression via the transcription factor FKHR (FOXO1). Gene Dev. 2004, 18(9): 1060-1071. Pizurki L, Zhou Z, Glynos K, et al. Angiopoietin-1 inhibits endothelial permeability, neutrophil adherence and IL-8 production. Brit J Pharmacol. 2003, 139(2): 329-336. Mesenchymal stem cells (MSC) nNo ethical difficulties nharvest MSC from the individual to be treated nNo rejection problems nSelf migrate to where the injury is located Rojas M, Xu J, Woods CR, et al. Bone marrow-derived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell Mol Biol 2005; 33: 145152 Robert JD， Annemarie BM mesenchymal stem cells： biology and potential clinical uses. Experimental Hematology, 2000, 28(8):87 Our hypothesis +Ang1 MSCs By Lentiviral vector MSCs transduced with Ang1 Mice LPS induced lung injury In vivo intervention Methods and results pCR2.1-Ang1 : Prof. Federico Bussolino in Institute for cancer research and treatment, Italy Sequencing results: 100% coincide with Ang1 cDNA sequence Electrophoresis after cutting by restriction enzyme pLV pCMV-dR8.74 pMD2G In vitro Lentivirus-mediated Ang1/eGFP transfer into MSCs Isolation, purification and expansion of MSCs n Bone marrow was harvested from 6 weeks old C57BL/6 by flushing the femurs and tibias n Separate MSCs from bone marrow by density gradient centrifugation in Ficoll (Pharmacia) n Cultured and passaged with Peisters method Peister A, Mellad JA, Larson BL, Hall BM, Gibson LF, Prockop DJ. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 2004; 103:1662-1668. MSCs transduced with Lenti- Ang1/eGFP n 1106 MSCs from passage 4 were seeded into a 12-well plate n Multiplicity of infection (MOI) : 10， 20， 40 ， 80 Merge photos Ang1 mRNA in MSCs M MSCs MSCs-Ang1 Ang1 protein in MSCs LPS induced ALI n 3 mg per ml LPS: E coli. Serotype 055: B5 n By aerosolization for 20 min using a flow rate of 6 liters/min n 7 successive days Ingenito EP, Mora R, Cullivan M, Marzan Y, Haley K, Mark L, and Sonna LA. Decreased surfactant protein- B expression and surfactant dysfunction in a murine model of acute lung injury. Am J Respir Cell Mol Biol 2001; 25: 35-44 Deliver MSCs to mice n By jugular vein injection n 1106 cells in 200l NS per mouse n Sacrifice mice and collect samples on day 0,3, 7, 14 after injection Ang1 protein in lungs * Quantitation of Ang1 protein * GFP was observed in lung by fluorescent microscope Fluorescent Light DAPI GFP(+)cells in