中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (6): 1061-1064.doi: 10.3969/j.issn.1673-8225.2011.06.024

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

中性粒细胞极性化过程中定向和侧足形成的方法学模式

陈海洋1,蔡春青1,谢翠华2,罗  琼1,孟晓静1   

  1. 1南方医科大学公共卫生与热带医学学院职业卫生与职业医学系,广东省广州市510515
    2南方医院内分泌代谢科,广东省广州市  510515
  • 收稿日期:2010-11-15 修回日期:2010-12-30 出版日期:2011-02-05 发布日期:2011-02-05
  • 通讯作者: 孟晓静,博士,教授,南方医科大学公共卫生与热带医学学院职业卫生与职业医学系,广东省广州市510515 zhumeng@fimmu.com
  • 作者简介:陈海洋★,男,1984年生,广东省高州市人,汉族,2011年南方医科大学毕业,硕士,主要从事细胞移动方面的研究。 ocean198511@sina.com 并列第一作者:蔡春青,男,1969年生,河南省焦作市人,汉族,2004年解放军第一军医大学毕业,博士,副教授,主要从事细胞电生理方面的研究。 h_zcn@126.com
  • 基金资助:

    国家自然科学基金(30971193,30800438 和81071611)。

Methodological study on directional sensing and lateral pseudopodia formation during neutrophil polarity

Chen Hai-yang1, Cai Chun-qing1, Xie Cui-hua2, Luo Qiong1, Meng Xiao-jing1   

  1. 1Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou  510515, Guangdong Province, China
    2Department of Endocrinology and Metabolism, Nanfang Hospital, Guangzhou  510515, Guangdong Province, China
  • Received:2010-11-15 Revised:2010-12-30 Online:2011-02-05 Published:2011-02-05
  • Contact: Meng Xiao-jing, Doctor, Professor, Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, Guangdong Province, China zhumeng@fimmu.com
  • About author:Chen Hai-yang★, Master, Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, Guangdong Province, China ocean198511@sina.com Cai Chun-qing, Doctor, Associate professor, Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, Guangdong Province, China h_zcn@126.com Chen Hai-yang and Cai Chun-qing contributed equally to this paper.
  • Supported by:

    the National Natural Science Foundation of China, No. 30971193*, 30800438*, 81071611*

摘要:

背景:中性粒细胞极性是其趋化和产生防御功能的基础,而极性化过程中定向能力和侧足形成是重要的环节,目前相关研究的报道还很少。
目的:观察均匀浓度、陡峭浓度梯度、平缓浓度梯度3种实验模式下细胞极性过程中片足的方向和侧足的形成情况,试图找出研究中性粒细胞极性的方法学模式。
方法:①采用密度梯度离心法,成功急性分离人中性粒细胞。②均匀浓度、陡峭浓度梯度和平缓浓度梯度甲酰甲硫氨酰-亮氨酰-苯丙氨酸对细胞进行趋化刺激。③倒置显微镜下观察细胞极性变化情况,判断细胞移动方向。④对细胞进行荧光双标,激光共聚焦显微镜下观察F-actin聚合情况。
结果与结论:①均匀浓度下,中性粒细胞极性化方向和移动轨迹随机分布。②陡峭梯度浓度下,41%的细胞呈现电极向心性分布。③平缓梯度下,68%的细胞表现为沿着甲酰甲硫氨酰-亮氨酰-苯丙氨酸浓度的分布,χ2检验提示平缓浓度梯度实验模式存在较好的定向性。④三者均有明显的侧足形成,以均匀浓度为高,这与定向性呈现为相反的结果。结果显示平缓浓度梯度实验模式在研究细胞定向机制方面相对较好,另一方面对细胞定向的干扰可能来源于细胞侧足的形成。

关键词: 嗜中性粒细胞, 极性, 侧足, N-甲酰-甲硫氨酰-亮氨酰苯丙氨酸, F-actin

Abstract:

BACKGROUND: Neutrophil chemotaxis is an important response in the host defense and initiated by neutrophil polarization. A key event of neutrophil polarity is the ability of directional sensing and lateral pseudopodium formation. However, there are few reports regarding the correlative investigation of the neutrophil polarization.
OBJECTIVE: To compare the three experimental modes of N-formyl-methionyl-leucyl-phenylalanine (Fmlp)-stimulated neutrophil polarity to provide some methodological considerations.
METHODS: ①Isolation of neutrophil by density gradient centrifugation; ②The neutrophil were stimulated by uniform concentration of fMLP, steep gradient concentration of fLMP and gentle gradient concentration of fLMP; ③The change and direction of neutrophil polarization were observed by inverted microscope; ④F-actin and cell nucleus were observed by confocal microscopy.
RESULTS AND CONCLUSION: ①The direction of neutronphil treated by uniform concentration of fMLP was random. ②41% of neutrophil migrated electrode-oriented when stimulated by steep gradient concentration of fLMP and 68% of neutrophil move toward the fMLP side upon stimulated by gentle gradient concentration of fLMP. ③χ2 test has been shown that there are significant difference in the polarized direction among neutrophils treated by three experimental modes. ④Neutronphil lateral pseudopodia formed in all of three experimental modes, especially in the group of uniform concentration. It is better to use zigmond chamber to study on the mechanism of neutrophils directional sensing, which could be interfered by the formation of neutrophil lateral pseudopodia.

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