Siglec-1在巨噬细胞吞噬脂质及分泌趋化因子中的作用  

The role of Siglec-1 in chemokines secretion and phagocytosis of oxidized low.density lipoprotein on macrophages

作　　者：熊怡淞[1] 李畅[1] 孙懿[1] 仲人前[1] 

机构地区：[1]第二军医大学长征医院实验诊断科全军医学免疫诊断中心全军临床免疫重点实验室,上海200003

出　　处：《中华检验医学杂志》2010年第7期691-696,共6页Chinese Journal of Laboratory Medicine

基　　金：国家863高科技研究发展计划资助项目（2006AA02Z496）;上海市科学技术委员会优秀学科带头人基金资助项目（07XD14013）

摘　　要：目的 研究ox-LDL刺激对体外培养小鼠巨噬细胞株RA-W264.7和原代小鼠骨髓巨噬细胞Siglec-1表达的影响,及M-CSF促进巨噬细胞Siglec-1高表达或小干扰RNA（si-RNP,）靶向抑制Siglec-1表达特点,并观察巨噬细胞吞噬脂质和分泌趋化因子的变化,以探讨Siglec-1在巨噬细胞参与AS中的作用.方法 用铜氧化法制备ox-LDL,根据预实验结果,选取ox-LDL 100μg/ml作为刺激物,在含巨噬细胞溶液中加入不同类型si-RNA或不同浓度M-CSF后,分为6组[正常对照组（仅加巨噬细胞）、ox-LDL 100μg/ml组、ox-LDL 100 μg/ml＋Si-RNA 25092 ng/ml组、ox-LDL 100μg/ml＋si-RNA 36182 ng/ml组、ox-LDL 100 μg/ml＋M-CSF 5 ng/ml组和ox-LDL 100μg/ml＋M-CSF 10 ng/ml 组].分别用si-RNA转染巨噬细胞抑制Siglec-1表达,M-CSF刺激促进巨噬细胞Siglec-1表达,并用荧光定量PCR检测Siglec-1抑制或表达增强效果.再用ox-LDL刺激48 h收集细胞和培养上清液,ELISA测定培养上清液中MIP-1 alpha、MCP-1和IL-8的浓度（每组3复孔）,以观察巨噬细胞活化情况;油红0染色观察Siglec-1抑制或表达增强后对巨噬细胞吞噬脂质颗粒的影响（每组3复孔）.结果 巨噬细胞经si-RNA转染后,用荧光显微镜观察,si-RNA能有效转染巨噬细胞,转染效率约90%;荧光定量PCR检测si-RNA的结果显示,当si-RNA 2509和si-RNA,3618在最佳转染浓度（40 pmol/L）时,对Siglec-1抑制率分别为0.54±0.11和0.52±0.16;比正常对照组（1.00±0.24）高（t值分别为5.227、4.992,P均〈0.01）.M-CSF 10.g/ml刺激48 h后Siglec-1 mRNA表达增加约4倍（4.16±1.25比1.00±0.24,t=7.448,P〈0.01）.si-RNA 3618干扰Siglec-1后,MCP-1、MIP-1alpha和NIP-23个巨噬细胞趋化因子的分泌分别为（359.28±47.80）pg/ml、（33.76±14.28）ng/ml、（7.87±1.55）ng/ml,比单纯加ox-LDL 100μg/ml组明显降低[分别为（577.89±35.95）pg/ml、（69.17±11.82）ng/ml、（12.28±1.19）ng/ml,P分别〈0.001、0.05、0.01];而加M-CSF 10 ng/ml组可明显促进巨�Objective By in vitro culture of mouse macrophage cell line RAW264. 7 and primary mouse bone marrow macrophages, the expression of Siglec-1 when stimulated by ox-LDL was observed. Meanwhile, Siglec-1 was up-regulated by M-CSF and down-regulated by small interference RNA targeting Siglec-1 （ si-RNA-Siglec-1） , and the expression of chemokines and lipid uptake ability by macrophages were observed, to explore the role of Siglec-1 on macrophages in atherosclerosis. Methods LDL was oxidized by copper. According to preliminary experiment results, ox-LDL 100 μg/ml was selected as a stimulus. There were 6 experimental groups：normal control group,ox-LDL 100 μg/ml group, ox-LDL 100 μg/ml ＋ si-RNA 2509 2 ng/ml group,ox-LDL 100 μg/ml ＋ si-RNA 3618 2 ng/ml group,ox-LDL 100 μg/ml ＋ M-CSF 5 ng/ml group and ox-LDL 100 μg/ml ＋ M-CSF 10 ng/ml group. si-RNA-Siglec-1 was transfected into macrophage to inhibit the expression of Siglec-1, whereas M-CSF 10 ng/ml or 5 ng/ml were added into the culture medium to enhance the expression of Siglec-1. Quantitative real-time polymerase chain reaction （ qRT-PCR） was used to determine the interfere efficiency of si-RNA-Siglec-1 or M-CSF. After stimulation with ox-LDL for 48 h, cell culture supernatants were collected to determine MIP-1 alpha, MCP-1 and IL-8 concentration by ELISA （n =3 for each group） to evaluate the activation of macrophages. Internalization of lipid particles by macrophages was analyzed by oil red 0 staining. Results Observed by fluorescence microscope, si-RNA-Siglec-1 could be effectively transfected into macrophages with a transfection efficiency about 90% ;PCR results showed that si-RNA 2509 and si-RNA 3618 in a concentration of 40 pmol/L had an inhibition rate of 0. 54 ±0. 11 or 0. 52 ±0. 16 vs 1. 00 ±0. 24 （control group） , t =5. 227 and 4. 992, respectively, all P 〈 0.01, while M-CSF 10 ng/ml could increase Siglec-1 mRNA expression approximately 4-fold （4. 16 ± 1. 25 vs 1.00 ±0. 24, t =7. 448, P〈0. 01）. The secretion of MCP-1

关 键 词：动脉粥样硬化 氧化型低密度脂蛋白 唾液酸结合的免疫球蛋白样凝集素-1 巨噬细胞 吞噬 趋化因子 

分 类 号：R392[医药卫生—免疫学]