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
간소통과Toll양수체4감소지다당자격인내피세포립세포집락자격인자적표체
Unfractionated heparin inhibits lipopolysaccharide-induced expression of granulocyte colony-stimulating factor in human endothelial cells through Toll-like receptor 4 signaling pathway
  • 万方数据
    中华危重病急救医学 중화위중병급구의학
    Chinese Critical Care Medicine
    2015年 2期 81-85 ,共5页

    李旭%刘一娜%马晓春

    리욱%류일나%마효춘

    脂多糖%内皮细胞%普通肝素%Toll样受体%粒细胞集落刺激因子 脂多糖%內皮細胞%普通肝素%Toll樣受體%粒細胞集落刺激因子
    지다당%내피세포%보통간소%Toll양수체%립세포집락자격인자
    Lipopolysaccharide%Endothelial cell%Unfractionated heparin%Toll-like receptor%Granulocyte colony-stimulating factor
    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+0.1 U/mL肝素组和LPS+1 U/mL肝素组4组，肝素作用组于LPS刺激前15 min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24 h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4 h加入5μg/mL球形红细菌LPS（LPS-RS，一种TLR4拮抗剂）；于LPS刺激24 h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+0.1 U/mL肝素组和LPS+1 U/mL肝素组，处理方法同实验1；于LPS刺激1 h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，0.1 U/mL和1 U/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＜0.05〕，两个肝素组间无明显差异，但以1 U/mL肝素作用较明显。② LPS-RS可明显抑制LPS诱导的IL-6、 G-CSF水平增高〔IL-6（ng/L）：139.1±37.6比655.9±58.3， G-CSF（ng/L）：73.7±19.7比388.7±36.2，均P＜0.05〕；而单独应用LPS-RS对细胞因子无明显影响〔IL-6（ng/L）：118.2±42.1比75.5±18.2，G-CSF （ng/L）：48.4±26.8比35.3±12.6，均P＞0.05〕。③ LPS刺激1 h后TLR4蛋白表达（灰度值）明显高于对照组（0.87±0.23比0.36±0.12，P＜0.05）；0.1 U/mL和1 U/mL肝素预处理均可明显抑制LPS诱导的TLR4蛋白表达（0.68±0.18、0.62±0.26比0.87±0.23，均P＜0.05）。结论 LPS刺激下HPMEC中IL-6、G-CSF表达增加，肝素可能通过调节TLR4降低其表达水平，从而发挥细胞保护作用。
    목적：관찰간소대지다당（LPS）자격인내피세포립세포집락자격인자（G-CSF）수평적영향，병탐토Toll양수체4（TLR4）재기과정중적가능작용。방법체외배양인폐미혈관내피세포（HPMEC），취전대배양지제3～5대세포용우실험。실험1：장세포분위대조조、LPS자격조（LPS 10μg/mL）、LPS+0.1 U/mL간소조화LPS+1 U/mL간소조4조，간소작용조우LPS자격전15 min가입상응제량보통간소，대조조가입여LPS등량적린산염완충액（PBS）；우LPS자격24 h후수집세포상청，채용매련면역흡부시험（ELISA）측정백세포개소-6（IL-6）、 G-CSF수평，이명학간소대HPMEC적작용。실험2：령취세포，분별우가입PBS혹LPS전4 h가입5μg/mL구형홍세균LPS（LPS-RS，일충TLR4길항제）；우LPS자격24 h후수집세포상청，측정IL-6、 G-CSF수평，이명학TLR4재LPS유도HPMEC손상중적작용。실험3：령취세포，분위대조조、LPS자격조、LPS+0.1 U/mL간소조화LPS+1 U/mL간소조，처리방법동실험1；우LPS자격1 h후수집세포，채용단백질면역인적시험（Western Blot）측정TLR4단백표체，이명학간소대TLR4적작용。결과①여대조조비교，LPS자격조IL-6급G-CSF수평명현증고〔IL-6（ng/L）：655.9±58.3비75.5±18.2，G-CSF（ng/L）：388.7±36.2비35.3±12.6，균P＜0.05〕；여LPS자격조비교，0.1 U/mL화1 U/mL간소예처리균가명현강저IL-6급G-CSF수평〔IL-6（ng/L）：518.2±64.6、489.1±75.6비655.9±58.3，G-CSF（ng/L）：298.8±41.0、273.4±33.2비388.7±36.2，균P＜0.05〕，량개간소조간무명현차이，단이1 U/mL간소작용교명현。② LPS-RS가명현억제LPS유도적IL-6、 G-CSF수평증고〔IL-6（ng/L）：139.1±37.6비655.9±58.3， G-CSF（ng/L）：73.7±19.7비388.7±36.2，균P＜0.05〕；이단독응용LPS-RS대세포인자무명현영향〔IL-6（ng/L）：118.2±42.1비75.5±18.2，G-CSF （ng/L）：48.4±26.8비35.3±12.6，균P＞0.05〕。③ LPS자격1 h후TLR4단백표체（회도치）명현고우대조조（0.87±0.23비0.36±0.12，P＜0.05）；0.1 U/mL화1 U/mL간소예처리균가명현억제LPS유도적TLR4단백표체（0.68±0.18、0.62±0.26비0.87±0.23，균P＜0.05）。결론 LPS자격하HPMEC중IL-6、G-CSF표체증가，간소가능통과조절TLR4강저기표체수평，종이발휘세포보호작용。
    ObjectiveTo 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 microvascular endothelial cells (HPMECs) were cultured in vitro, and the cells between passages 3 and 5 were used in the experiments. ExperimentⅠ: the cells were divided into four groups as follows: control group, LPS stimulation group (LPS 10μg/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. ExperimentⅡ: HPMECs were treated with 5μg/mL of rhodobacter sphaeroides LPS (LPS-RS,&nbsp;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. ExperimentⅢ: 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 experimentⅠ. 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.Results① 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+ 1 U/mL UFH group, the levels of IL-6 and G-CSF were significantly decreased [IL-6 (ng/L): 518.2±64.6, 489.1±75.6 vs. 655.9±58.3, G-CSF (ng/L): 298.8±41.0, 273.4±33.2 vs. 388.7±36.2, allP< 0.05]. The results indicated that 1 U/mL UFH had better results, though there was no statistical significance between the results of two UFH groups.② LPS-induced up-regulation of IL-6 and G-CSF levels was prevented by LPS-RS [IL-6 (ng/L): 139.1±37.6 vs. 655.9±58.3, G-CSF (ng/L): 73.7±19.7 vs. 388.7±36.2, bothP< 0.05]. LPS-RS alone had no effect on cytokines [IL-6 (ng/L):118.2±42.1 vs. 75.5±18.2, G-CSF (ng/L): 48.4±26.8 vs. 35.3±12.6, bothP> 0.05].③ Compared with control group, the protein expression of TLR4 (grey value) in LPS stimulation group was significantly upregulated after 1 hour (0.87±0.23 vs. 0.36±0.12,P< 0.05). UFH with 0.1 U/mL and 1 U/mL lowered TLR-4 protein expression induced by LPS (0.68±0.18, 0.62±0.26 vs. 0.87±0.23, bothP< 0.05).ConclusionsThe expressions of IL-6 and G-CSF were increased obviously in LPS treated HPMECs. UFH might take its therapeutic effect through TLR4-dependent pathway.
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