高表达Ang1基因的间充质干细胞株的建立.pptVIP

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2016-09-14 发布于重庆
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高表达Ang1基因的间充质干细胞株的建立.ppt

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高表达Ang1基因的间充质干细胞株的建立

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 endothelial cells Mesenchymal stem cells (MSC) No ethical difficulties harvest MSC from the individual to be treated No rejection problems Self migrate to where the injury is located Methods and results pCR2.1-Ang1 : Prof. Federico Bussolino in Institute for cancer research and treatment, Italy Electrophoresis after cutting by restriction enzyme 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 MSCs from bone marrow by density gradient centrifugation in Ficoll (Pharmacia) Cultured and passaged with Peister’s method MSCs transduced with Lenti-Ang1/eGFP 1×106 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 Ang1 protein in MSCs LPS induced 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 Deliver MSCs to mice By jugular vein injection 1×106 cells in 200μl NS per mouse Sacrifice mice and collect samples on day 0,3, 7, 14 after injection Ang1 protein in lungs GFP was observed in lung by fluorescent microscope 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 This is the Icon of the plasmid contained Ang1 pLV，pCMV-dR8.74 and pMD2G co-transfer light jugular [dZˉgjulE] 颈的,颈静脉的 * * 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, Burov