肝素通过Toll样受体4减少脂多糖刺激人内皮细胞粒细胞集落刺激因子的表达  

Unfractionated heparin inhibits lipopolysaccharide-induced expression of granulocyte colony-stimulating factor in human endothelial cells through Toll-like receptor 4 signaling pathway

作　　者：李旭[1] 刘一娜[1] 马晓春[1] 

机构地区：[1]中国医科大学附属第一医院重症医学科,辽宁沈阳110001

出　　处：《中华危重病急救医学》2015年第2期81-85,共5页Chinese Critical Care Medicine

基　　金：基金项目：国家自然科学基金（81101411）;辽宁省自然科学基金（2013021061）

摘　　要：目的 观察肝素对脂多糖（LPS）刺激人内皮细胞粒细胞集落刺激因子（G—CSF）水平的影响，并探讨Toll样受体4（TLR4）在其过程中的可能作用。方法 体外培养人肺微血管内皮细胞（HPMEC），取传代培养至第3。5代细胞用于实验。实验1：将细胞分为对照组、LPS刺激组（LPS 10μg／mL）、LPS＋0．1U／mL肝素组和LPS＋1U／mL肝素组4组，肝素作用组于LPS刺激前15min加入相应剂量普通肝素，对照组加入与LPS等量的磷酸盐缓冲液（PBS）；于LPS刺激24h后收集细胞上清，采用酶联免疫吸附试验（ELISA）测定白细胞介素-6（IL-6）、G—CSF水平，以明确肝素对HPMEC的作用。实验2：另取细胞，分别于加入PBS或LPS前4h加入5μg／mL球形红细菌LPS（LPS—RS，一种TLR4拮抗剂）；于LPS刺激24h后收集细胞上清，测定IL-6、G—CSF水平，以明确TLR4在LPS诱导HPMEC损伤中的作用。实验3：另取细胞，分为对照组、LPS刺激组、LPS＋0．1U／mL肝素组和LPS＋1U／mL肝素组，处理方法同实验1；于LPS刺激1h后收集细胞，采用蛋白质免疫印迹试验（Western Blot）测定TLR4蛋白表达，以明确肝素对TLR4的作用。结果 ①与对照组比较，LPS刺激组IL-6及G—CSF水平明显增高[IL-6（ng／L）：655．9±58．3比75．5±18．2，G-CSF（ng／L）：388．7±36．2比35．3±12．6，均P〈0．05]；与LPS刺激组比较，0．1U／mL和lU／mL肝素预处理均可明显降低IL-6及G—CSF水-平（IL-6（ng／L）：518．2±64．6、489．1±75．6比655．9±58．3，G—CSF（ng／L）：298．8±41．0、273．4±33．2比388．7±36．2，均P〈O．05]，两个肝素组间无明显差异，但以1U／mL肝素作用较明显。②LPS—RS可明显抑制LPS诱导的IL-6、G-CSF水平增高[IL一6（n∥L）：139．1±37．6比655．9±58-3，G—CSF（ng／L）：73．7±19．7比388．7±36．2，均JP〈0．05]；而单独应用LPS—RS对细胞因子无明显影响[IL一6（ng／LObjective To determine the effect of unfractionated heparin （UFH） on lipopolysaccharide （LPS）- induced expression of granulocyte colony-stimulating factor （ G-CSF ）, and the role of Toll-like receptor 4 （ TLR4 ） signaling pathway in this process. Methods Human pulmonary microvaseular endothelial cells （HPMECs） were cultured in vitro, and the ceils between passages 3 and 5 were used in the experiments. Experiment I ： the cells were divided into four groups as follows： control group, LPS stimulation group （LPS 10 μg/mL）, LPS ＋ 0.1 U/mL UFH group, and LPS ＋ 1 U/mL UFH group. HPMECs in UFH groups were treated with 0.1 U/mL or 1 U/mL UFH 15 minutes before LPS stimulation, and HPMECs in control group were treated with an equal volume of phosphate- buffered saline （PBS） instead. The concentrations of interleukin-6 （IL-6） and G-CSF in cell culture supernatants were determined by enzyme linked immunosorbent assay （ELISA） 24 hours after LPS challenge to detect the effect of UFH on HPMECs. Experiment lI ： HPMECs were treated with 5 μg/mL of rhodobacter sphaeroides LPS （LPS-RS,antagonist for TLR4） 4 hours before the addition of PBS or LPS. The concentrations of IL-6 and G-CSF in cell culture supernatants were determined 24 hours after LPS stimulation to detect the effect of TLR4 on LPS-induced HPMEC injury. Experiment m ： HPMECs were divided into four groups as before： control group, LPS stimulation group, LPS ＋ 0.1 U/mL UFH group, LPS ＋ 1 U/mL UFH group. Treatments to cells were the same as experiment I . The protein expression of TLR4 in HPMECs was determined by Western Blot 1 hour after LPS stimulation to detect the effect of UFH on TLR4. Results ① Compared with control group, the levels of IL-6 and G-CSF in LPS stimulation group were increased [ IL-6 （ng/L）： 655.9 ± 58.3 vs. 75.5 ± 18.2, G-CSF （ng/L）： 388.7± 36.2 vs. 35.3±12.6, both P 〈 0.05 ]. Compared with those of LPS stimulation group, in LPS ＋ 0.1 U/mL UFH group and LPS ＋ I U/mL UFH

关 键 词：脂多糖 内皮细胞 普通肝素 TOLL样受体 粒细胞集落刺激因子 

分 类 号：R96[医药卫生—药理学]