The Role Of β2-GlycoproteinⅠin The Clearance Of Apoptotic HL-60Cells By THP-1Derived Phagocyte

Objective:To investigate the role of β2-glycoprotein Ⅰin the clearance of apoptoticHL-60cells by THP-1derived phagocytes and try to explore its mechanism.Methods:HL-60cells in vitro were treated with cytosine arabinoside(Ara-C) and detected cellgrowth inhibition rate and apoptosis rate with MTT method and AnnexinV-PI methodrespectively.PMA was used to induce THP-1cells differentiating into phagocytic cells.Verification of THP-1phagocytic phenotypic was done by CD11b.The phagocytic capacitywas detected by ink phagocytosis experiment. Binding specifity of β2-gp1to PS wastested in ELISA. AnnexinV/β2-gp1competition experiments were performed to detect thebinding of β2-gp1to phosphatidylserine on the apoptotic cells. And we discussed the roleof β2-gp1in phagocytosis of apoptotic cells.Results:1.The apoptosis of HL-60cells:HL-60cells which were treated with a serialconcentration of Ara-C after24and48hours appeared typical changes in morphology：the cell size became smaller, nuclear shrinkage or splitting,chromatin concentrated oredged. The cell growth inhibition rate and apoptosis rate were increased with theconcentration of Ara-C rising.24hours after800ug/ml Ara-C on HL-60cells,the cellgrowth inhibition rate was up to72.50±1.64%,the early and late apoptotic rate were19.35±0.44%and53.56±2.90%,respectively which were significantly higher than the controlgroup.2.Differentiation of THP-1and verification of phagocytic phenotypic andphagocytic capacity:When THP-1cells were treated with150ng/ml PMA for72hours, the morphology of THP-1cells showed significant changes: cell size bigger, cell shape irregular andStretched out pseudopodia. The expression of CD11b on the cell surface were17.71±0.49%,18.77±0.66%,22.93±0.53%,21.29±0.87%at the concentration of50,100,150,200ng/ml PMA treating48hours, respectively. After72hours of aboveconcentration of PMA, the positive rate of CD11b in each group was43.89±1.64%,49.14±0.72%,54.88±1.73%,52.11±0.81%.At the same concentration of PMA, the positiverate of CD11b in72hours group was significantly higher than in the48hours group（P<0.05）.When THP-1cells were treated with150ng/ml PMAfor72hours, the ability ofswallow ink was up to90%or more.3.The binding specificity of β2-gpⅠto phosphatidylserine:The result of ELISA showed positive which prompted that β2-gpⅠcan bind to PS.4.β2-gpⅠ/AnnexinV competition experiments:The results of flow cytometry showed that the binding rates of AnnexinV andapoptotic cells were gradually decreased with the increasing concentration of β2-gpⅠ.The binding rate of AnnexinV and apoptotic cells was22.72%without β2-gpⅠ.Whilethe binding rate were21.43%,14.00%,13.15%by adding100ng/ul,200ng/ul,400ng/ulβ2-gpⅠwhich were significantly lower than the group without β2-gpⅠ(P<0.05).And thebinding rate of β2-gpⅠand early apoptotic cells was decreased with time.5.The effect of β2-gpⅠon the phagocytosis of apoptotic cells:The phagocytosis of apoptotic HL-60cells was10.5%without β2-gp Ⅰ.Thephagocytosis became16.8%、33.6%、36.83%when the concentration of β2-gpⅠwas50ug/ml、100ug/ml、150ug/ml. And the phagocytosis were increased with the increasingconcentration of β2-gpⅠ.Conclusion:β2-gpⅠ can bind to apoptotic HL-60cells and enhance the phagocytosis of theapoptotic cells.