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
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