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| CD31 a Specific Marker: Purification of Porcine Liver Sinusoidal Endothelial Cells |
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Abstract Platelet endothelial cell adhesion molecule-1 (CD31) is an important endothelial cell surface membrane molecules, which has been used to identify endothelial cells. However, whether CD31 can be used as specific marker of purified pig (Sus scrofa) sinusoidal endothelial cells (porcine liver sinusoidal endothelial cell, pLSEC) should be verified. Therefore, in this study, whether purified pLSEC had iconic aperture structure were observed by flow sorting separation pLSEC with CD31 marker. α-1,3-galactosidase transferase gene knockout (GTKO) of Wuzhishan miniature pigs were used as experiment material. These methods including liver perfusion in situ collagenase digestion in vitro, percoll selective gradient centrifugation and adherent culture, fluorescein isothiocyanate-CD31(FITC-CD31) marker pLSEC streaming sorting were adopted. The results of observations of endothelial cells of isolated pLSEC by inverted microscope showed that typical cell morphology, cobblestone neatly arranged, TEM cells ultrastructure fenestrae. Cells could be observed to have fenestrations through environmental scanning electron microscope,cell surface structure of pLSEC and porcine aortic endothelial cells were obvious different from each other which could be observed through transmission electron microscope, pLSEC had a special aperture structure. qRT-PCR was used to detect CD31, cell adhesion molecules (CD146), hemophilia factor Ⅷ gene (Ⅷ-F) and von willebrand factor gene (vWF) of pLSEC, pAEC, porcine ear fibroblast cell (pEFC). The results of expression showed that pLSEC of CD31, CD146, Ⅷ factor and vWF expression levels have were higher than pAEC and pEFC significantly. Then phagocytosis was detected using immunofluorescence pLSEC which had an innered cell membrane fluorescent probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate acetylated low density lipoprotein (DiI-Ac-LDL) uptake. In a word, CD31 can be used as a special marker to purify pLSEC, which provides an effective way for the separation of liver sinusoidal endothelial cells.
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Received: 11 April 2016
Published: 01 July 2016
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| [1]Albelda S M, Muller W A, Buck C A, et al.Molecular and cellular properties of PECAM-1 (endoCAMCD31): a novel vascular cell-cell adhesion molecule[J].Journal of Cell Biology, 1991, 114(5):1059-68
[2]Dian F, Nagy J A, Kathryn P, et al.Ultrastructural localization of platelet endothelial cell adhesion molecule (PECAM-1,CD31) in vascular endothelium[J].Journal of Histochemistry & Cytochemistry, 2004, 52(1):87-101
[3]Lollar J S, Do H V, Healey J F, et al.Compositions and methods for the expression of factor VIII polypeptides and uses therefor: US, US 20030129174 A1[P]. 2003.
[4]Lollar J S, Do H V, Healey J F, et al.Compositions and methods for the expression of factor VIII polypeptides and uses therefor: US, US 20030129174 A1[P]. 2003.
[5]赵秀华, 罗彬, 罗盘, 程腊梅.小鼠肝脏窦状内皮细胞分离和培养的一种新方法[J]., 2010, 30(1):1-7
[6]Xiu-Hua Zhao, Bing Luo, Pan Luo, Cheng Lamei.Shape of mouse liver sinus endothelial cell isolation and culture of a kind of new method [J]. International journal of pathology and clinical science, 2010, 01:1-7.[J].international joumal of pathology and clinical medicine, 2010, 30(1):1-7
[7]Deleve LD, Wang X, Mccuskey MK et al.Rat liver endothelial cells isolated by anti-CD31 immunomagnetic separation lack fenestrae and sieve plates[J].Am J Physiol Gastrointest Liver Physiol, 2006, 291(6):G1187-9
[8]王飞.GGAT1基因敲除猪的制备及其功能验证[D].四川农业大学, 2013.
[9]Fei Wang.Preparation and it is functional verification of GGAT1 knockout pigs [D]. Sichuan agricultural university, 2013.
[10]Livak K J, Schmittgen T D.Analysis of relative gene expression data using Real-time quantitative PCR and the 2(-Delta C(T)) method[J]. Methods, 25(4): 402-408.[J]., 2001, 25(14):402-408
[11]Paris LL, Chihara RK, Reyes LM et al.ASGR1 expressed by porcine enriched liver sinusoidal endothelial cells mediates human platelet phagocytosis in vitro. Xenotransplantation 2011; 18: 245–251.[J]., 2011, 18(4):245-251
[12]Chihara RK, Paris LL, Reyes LM et al.Primary por-cine Kupffer cell phagocytosis of human platelets involves the CD18 receptor. Transplantation 2011; 92: 739–744.[J]., 2011, 92(7):739-744
[13]Paris LL, Chihara RK, Sidner RA, Tector AJ, Burlak C.Differences in human and porcine platelet ol-igosaccharides may influence phagocytosis by liver sinu-soidal cells in vitro. Xenotransplantation 2012; 19: 31–39.[J]., 2012, 19(1):31-39
[14]Burlak C, Paris LL, Chihara RK et al.The fate ofhuman platelets perfused through the pig liver: implica-tions for xenotransplantation. Xenotransplantation 2010; 17: 350–361.[J]., 2010, 17(5):350-361
[15]Ekser B, Long C, Echeverri GJ et al.Impact of thrombocytopenia on survival of baboons with genetically modi?ed pig liver transplants: clinical relevance[J]. Am J Transplant, 2010; 10: 273-285.[J]., 2010, 10(2):273-285
[16]Tector AJ, Fridell JA, Ellas N et al.Aberrations in hemostasis and coagulation in untreated discordant hepatic xenotransplantation: Lalor P F,Philip S,Allisterj G,et alRecruitment of lymphocytes to the human liver.[J].Immunology & Cell Biology, 2002, 80(1):52-64
[17]Deleve L D, Wang X, Kuhlenkamp J F, et al.Toxicity of azathioprine and monocrotaline in murine sinusoidal endothelial cells and hepatocytes: The role of glutathione and relevance to hepatic venoocclusive disease[J].Hepatology, 1996, 23(3):589-99
[18]Nedredal G I, Elvevold K H, Ytreb L M, et al.Liver sinusoidal endothelial cells represents an important blood clearance system in pigs[J].Comparative Hepatology, 2003, 2(Suppl 1):1-3
[19]Qiang P, Yeh H, Wei L, et al.Mechanisms of Xenogeneic Baboon Platelet Aggregation and Phagocytosis by Porcine Liver Sinusoidal Endothelial Cells[J].Plos One, 2012, 7(10):7-10
[20]Wang Z Y, Paris L L, Chihara R K, et al.Immortalized porcine liver sinusoidal endothelial cells: an in vitro model of xenotransplantation-induced thrombocytopenia[J].Xenotransplantation, 2012, 19(4):249-255
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