刘雪云, 刘永, 李金凤, 岳少杰, 申丽, 李晨, 汉建忠, 许建平, 冯丹丹, 刘惠君, 罗自强
中南大学湘雅医学院生理学系，长沙 410078； 福建中医药大学中西医结合系生理教研室，福州 350108； 中南大学湘雅医院儿科，长沙 410078
本文旨在探讨I 组代谢型谷氨酸受体(group I metabotropic glutamate receptor, mGluRI)在中性粒细胞上的表达，以及该受体的激活对中性粒细胞与内皮细胞相互黏附的影响。取健康人新鲜静脉血，Ficoll-Hypaque 密度梯度离心法分离中性粒细胞，免疫细胞化学法和real-time PCR 法检测中性粒细胞mGluRI (包括mGluR1 和mGluR5)的表达，分别应用不同浓度的mGluRI 特异性激动剂S-3,5- 二羟基苯甘氨酸(S-3,5-dihydroxy-phenylglycine, S-DHPG)处理中性粒细胞不同时间，通过比色法检测中性粒细胞和人正常脐静脉内皮细胞(human normal umbilical vein endothelial cells, HUVE-12)黏附率，采用流式细胞术测定中性粒细胞黏附分子CD11a 表达的变化。结果证实中性粒细胞表达mGluRI (mGluR1/5)；S-DHPG在1×10-8~1×10-6 mol/L浓度范围内呈剂量依赖性地提高中性粒细胞与内皮细胞之间的黏附率(P<0.05 或P<0.01)；1×10-6 mol/L S-DHPG 单独作用于中性粒细胞0.5 h，即可促进中性粒细胞黏附于内皮细胞(P<0.01) ，但没有时间依赖性；1×10-6 mol/L S-DHPG 单独作用于中性粒细胞可促进CD11a 表达(P<0.01)；mGluRI 拮抗剂(RS)-α- 甲基-4- 羧基苯甘氨酸[(RS)-α-methyl-4-carboxyphenylglycine,(±)-MCPG] (0.5 mmol/L)可以显著阻断激动剂S-DHPG (1×10-6 mol/L, 1 h)的促黏附效应(P<0.01)。上述结果证实了中性粒细胞膜上mGluRI 的激活可增强中性粒细胞表面黏附分子CD11a 的表达，促进中性粒细胞与内皮细胞的黏附。
[Activation of mGluRI in neutrophils promotes the adherence of neutrophils to endothelial cells.] [Ariticle in Chinese]
LIU Xue-Yun, LIU Yong, LI Jin-Feng, YUE Shao-Jie, SHEN Li, LI Chen, HAN Jian-Zhong, XU Jian-Ping, FENG Dan-Dan, LIU Hui-Jun, LUO Zi-Qiang
Department of Physiology, Xiangya School of Medicine, Central South University, Changsha 410078, China； Fujian University of Traditional Chinese Medicine, Fuzhou 350108, China； Department of Pediatrics, Xiangya Hospital, Central South University, Changsha 410078, China
L-glutamate (Glu) is an excitatory neurotransmitter in the mammalian central nervous system. Relatively much attention hasbeen paid to functional expression of Glu signaling molecules in peripheral tissues very recently. The present study tested thehypothesis that the activation of group I metabotropic glutamate receptor (mGluRI) in neutrophils stimulated neutrophils adherenceto endothelial cells by increasing the surface expression of certain adhesion molecules. Peripheral blood was obtained by venipuncturefrom healthy donors, and the neutrophils were isolated by Ficoll-Hypaque gradient centrifugation. Neutrophils floating into DMEM/F12 culture medium containing 10% fetal bovine serum were then used immediately. Immunocytochemistry and real-time quantitativeRT-PCR were used to detect the expression of mGluRI (mGluR1 and mGluR5) in neutrophils. The adherence of neutrophils to culturedhuman normal umbilical vein endothelial cells (HUVE-12) was measured by the colorimetric method. Cell surface expression ofadhesion molecule CD11a in the neutrophils was determined by flow cytometry. Immunocytochemistry and real-time quantitative RTPCRshowed that mGluR1 and mGluR5 were constitutively expressed in neutrophils. Application of mGluRI agonist S-3,5- dihydroxyphenylglycine (S-DHPG) (1×10-8-1×10-6 mol/L) showed a dose-dependent stimulatory effect on the adherence of neutrophilsto HUVE-12 (P<0.05 or P<0.01), with a maximum effect at 1×10-6 mol/L (P<0.01). Incubations as short as 30 min were sufficientto induce increased adherence after the beginning of S-DHPG treatment. Following time extension (0.5-5 h), S-DHPG (1×10-6 mol/L)increased the rate of neutrophils adhesion to HUVE-12 with a maximum effect at 0.5 h (P<0.01). However, a time-dependent effect ofS-DHPG on the rate of neutrophils adhesion to HUVE-12 was not observed during the experimental period. 1×10-6 mol/L of S-DHPGalso induced an increased surface expression of adhesion molecule CD11a (P<0.01) when neutrophils were preincubated with 1×10-6mol/L of S-DHPG for 1 h. Furthermore, the specific mGluRI antagonist (RS)-α-methyl-4-carboxyphenylglycine ((±)-MCPG, 0.5mmol/L) significantly abolished the stimulatory effect of S-DHPG (1×10-6 mol/L) on the adherence of neutrophils to HUVE-12 (P<0.01).These results suggest that the activation of mGluRI in neutrophils results in increased adhesion molecule CD11a expression andthereby promotes the adherence of neutrophils to endothelial cells.
通讯作者：罗自强 E-mail: firstname.lastname@example.org
刘雪云, 刘永, 李金凤, 岳少杰, 申丽, 李晨, 汉建忠, 许建平, 冯丹丹, 刘惠君, 罗自强. 中性粒细胞mGluRI 的激活促进其与内皮细胞相互黏附[J]. 生理学报 2010; 62 (3): 219-224.
LIU Xue-Yun, LIU Yong, LI Jin-Feng, YUE Shao-Jie, SHEN Li, LI Chen, HAN Jian-Zhong, XU Jian-Ping, FENG Dan-Dan, LIU Hui-Jun, LUO Zi-Qiang. [Activation of mGluRI in neutrophils promotes the adherence of neutrophils to endothelial cells.] [Ariticle in Chinese] . Acta Physiol Sin 2010; 62 (3): 219-224 (in Chinese with English abstract).