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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