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采用重复密度梯度离心的方法分离中性粒细胞,并对其在10～100 nmol/L fMLP不同浓度趋化诱导下产生极性化的极性化率和伪足长度变化趋势进行分析。结果表明在加入fMLP的短时间内,细胞都表现出极性化率明显上升的趋势,以100 nmol/L为最快,但在3～4 min内都会达到90%以上的极性化水平。同时,细胞伪足长度明显受到不同fMLP浓度刺激的影响,表现为伸展和回缩相的交替,从而组成一个振荡周期。组成振荡周期的伸展相和回缩相及振荡频率都明显受到不同fMLP浓度刺激的影响,依据伪足变化率本文提出了细胞极性活跃程度的分类。由于伪足的变化与F-actin的聚合密切相关,而F-actin的聚合又受到极性信号分子的调节,因此对伪足变化的分析有助于了解中性粒细胞极性信号传导通路的调节机制。
Neutrophils were isolated by repeated density gradient centrifugation. The changes of polarization and pseudopod length were analyzed under different concentrations of 10 ~ 100 nmol / L fMLP. The results showed that in the short time after the addition of fMLP, the cells showed the tendency of polarization increased significantly, with 100 nmol / L as the fastest, but within 3 ~ 4 min will reach more than 90% of the polarization. At the same time, the length of cell pseudopods was significantly affected by the stimulation of different concentrations of fMLP, manifested by the alternation of stretching and retracting phases, thus forming an oscillating period. The stretching phase, the contracting phase and the oscillating frequency of the oscillating period are obviously affected by the stimulation of different concentrations of fMLP. According to the changing rate of pseudopods, the classification of the degree of cellular polarity is proposed. Because the change of pseudopod is closely related to the polymerization of F-actin, and the polymerization of F-actin is regulated by polar signal molecules, the analysis of pseudopod variation helps to understand the relationship between the Regulatory mechanism.