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Experimental Research on Expression of hAT-Ⅲ Gene after Transfecting Vascular Endothelial-like Cells

Author: LiuXiaoZuo
Tutor: RenHua
School: Peking Union Medical College , China
Course: Science in cardiothoracic surgery
Keywords: mesenchymal stem cells vascular endothelium-like cells bone marrow vascular endothelial-like cells anticoagulant gene gene expression vascular endothelial-like cells human AT—III gene modified
CLC: R543
Type: Master's thesis
Year: 2007
Downloads: 38
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Abstract

PartⅠ:Experimental study on bone marrow mesenchymal stem cells orientiatedly induced and differentiated into endothelium-like cells[Objective]To investigate the possibility of BMMSCs as seed cells for tissue engineering by harvesting and isolating bone marrow mesenchymal stem cells（BMMSCs）,then inducing and differentiating them into endothelium-like cells in vitro.[Methods]1.Isolation,culture and proliferation of BMMSCs:Bone marrow mononuclear cells,which obtained sterily by Ficoll-Paque density gradient centrifugation and adherent culture,were cultured and expanded in vitro.2.BMMSCs differentiated into endothelium-like cells:BMMSCs were treated in medium with 10ng/ml VEGF for 21 days.[Results]1.Characterization of BMMSCs derived from bone marrowA total of 5ml bone marrow was obtained from the rib of each adult（n=5 donors）.After 5 passages of culture in vitro,approximately 4×10⁸ BMMSCs were obtained from each sample,and after 10 passages,about 8×10¹⁰ cells. BMMSCs expressed higher level of CD105、CD44、CD29 and lower level of KDR.2.Differentiation of BMMSCs into vascular endothelium-like cellsExpression of VWF was highest after 14 days and maintained afterwards. A layer of sub-confluent cells exhibited a Cobblestone-like morphology. Swallow-viscles were observed within differentiated cells by transmission electron microscope.Culture of endothelium-like ceils in extracellular matrix gel（ECMgel） resulted in vascular microtube formation within 5 days.[Conclusion]1.Enough and purified BMMSCs were able to be obtained by the methods of density gradient centrifugation and adherent culture. 2.BMMSCs can be differentiated into cells with characteristics of endothelial cells and be served as seed cells of tissue engineering blood vessel. PartⅡ:A comparison of the expression in anticoagulant factors between vascular endothelial-like cells' dissertation">vascular endothelial-like cells derived from BMMSCs and HUVEC[Objective]To observe the difference in anticoagulant ability of endothelial cells from different sources.[Methods]1.Human bone marrow mesenchymal stem cells（BMMSCs） were cultured,purified, and expanded by Fieoll-Paque density gradient centrifugation and adherent culture in vitro.Then they were induced and differentiated into vascular endothelial-like cells（VELCs） in medium with 10ng/ml VEGF.2.Human umbilical vein endothelial cells（HUVECs） were harvested by collagenase digest and cultured in vitro.3.The major anticoagulant gene expression of the VELCs and the HUVECs was detected and compared by RT-PCR.[Results]1.BMMSCs can be successfully differentiated into vascular endothelial-like cells in vitro,but they can not express mRNA of the major anticoagulant gene.2.HUVECs can express the mRNA of these genes.[Conclusion]BMMSCs can be differentiated into vascular endothelial-like cells in vitro, but their anticoagulant ability is inferior to HUVECs. PartⅢ:Experimental Research on Expression of hAT-ⅢGene after Transfecting Vascular Endothelial-like Cells[Objective]To provide experimental foundation for improving anticoagulant ability of tissue engineering vascular by transfecting AT-Ⅲgene into vascular endothelial-like cells derived from BMMSCs and observing expression of it.[Methods]1.Plasmid pBLAST49-hAT3 with human AT-Ⅲgene were transfected into vascular endothelial-like cells by liposome mediate;2.The AT-Ⅲexpression was determined at mRNA level and protein level by RT-PCR,IHC,Westen-blot and its activity.[Results]1.The VELCs can express AT-Ⅲgene mRNA at 72 hours and 96 hours after pBLAST49-hAT3 weretransfectedinto VELCs;2.The IHC result demonstrates that the AT-Ⅲexpression of VELCs was positive at 72 hours and 96 hours after pBLAST49-hAT3 weretransfectedinto VELCs;3.The Westen-blot result demonstrates that the AT-Ⅲexpression of VELCs culture fluid was positive at 72 hours and 96 hours after pBLAST49-hAT3 were transfected into VELCs;4.The AT-Ⅲactivity of VELCs culture fluid was low after pBLAST49-hAT3 were transfected into VELCs.[Conclusion]1.Plasmid pBLAST49-hAT3 with human AT-Ⅲgene can be transfected into VELCs successfully,and AT-Ⅲgene can be expressed by VELCs after transfection;2.VELCs can be improved its anticoagulant ability by gene modified.

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CLC: > Medicine, health > Internal Medicine > Heart, blood vessels ( circulatory ) disease > Vascular disease
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