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1、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,Ang1 decreased in LPS induced lung injury anti-inflammation: inhibits endothelial permeability, neutrophil adherence and IL-8 production Anti-apoptosis role to endothel
2、ial 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
3、. 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),No ethical difficulties harvest MSC from the individual to be treated No rej
4、ection problems Self 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 BMmesenchymal stem cells：biology and potential clinical uses. Experimen
5、tal 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
6、 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,Bone marrow was harvested from 6 weeks old C57BL/6 by flushing the femurs and tibias Separate MSC
7、s from bone marrow by density gradient centrifugation in Ficoll (Pharmacia) 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, ra
8、tes of proliferation, and differentiation potential. Blood 2004; 103:1662-1668.,MSCs transduced with Lenti-Ang1/eGFP,1106 MSCs from passage 4 were seeded into a 12-well plate Multiplicity of infection (MOI) : 10，20，40 ，80,Merge photos,Ang1 mRNA in MSCs,M MSCs MSCs-Ang1,Ang1 protein in MSCs,LPS induc
9、ed ALI,3 mg per ml LPS: E coli. Serotype 055: B5 By aerosolization for 20 min using a flow rate of 6 liters/min 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
10、lung injury. Am J Respir Cell Mol Biol 2001; 25: 35-44,Deliver MSCs to mice,By jugular vein injection 1106 cells in 200l NS per mouse 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 lung by immunohistochemistry stain,Contrast in different injured area,结论,高表达目的基因Ang1的MSCs可通过基因技术有效构建，移植体内后，可在肺内检测到MSCs的转化修复 MSCs可作为良好的基因治疗运载工具,Thank you,