【摘要】 目的观察体外培养的肺微血管内皮细胞(PMVEC)经全氟异丁烯(PFIB)染毒后其肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、细胞间黏附分子1(ICAM-1)、基质金属蛋白酶2(MMP-2)和MMP-9的含量变化。方法采用改进的组织块种植法分离培养大鼠PMVEC,细胞经鉴定后分为正常对照组和5个PFIB染毒组(n=3)。正常对照组为静息状态细胞;染毒组行PFIB暴露(浓度200 mg·m-3,持续5 min)染毒后0.5,1,2,4和8 h分别收集培养上清液和细胞裂解液。ELISA检测TNF-α,IL-1β,ICAM-1,MMP-2和MMP-9含量,明胶酶谱法分析MMP-2和MMP-9酶活性。结果 1依据细胞生长的形态特征、特异性抗原的表达和植物凝集素结合实验鉴定,采用肺组织块原代培养法获得的细胞均符合PMVEC的特征。2与正常对照组比较,PMVEC经PFIB染毒后2 h细胞裂解液中TNF-α含量显著升高(P<0.05),4和8 h明显回落。细胞培养上清液中TNF-α含量变化趋势与细胞裂解液中TNF-α变化趋势类似,仅发生时间稍滞后。3 PFIB染毒后PMVEC激活并合成IL-1β的时间稍滞后于TNF-α,但达峰与持续时间与TNF-α相似。4 PFIB染毒后PMVEC培养上清液中ICAM-1含量持续保持于较低水平。5 PFIB染毒组细胞裂解液中未检测到MMP-2活性,培养上清液中检测到较高水平的MMP-2含量与活性。PFIB染毒组的培养上清液和细胞裂解液中均未检测到MMP-9表达与活性。结论 PFIB染毒刺激存活的PMVEC合成并释放大量TNF-α,IL-1β,MMP-2和结合型ICAM-1。更多还原

【Abstract】 OBJECTIVE To investigate the change of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),intercel ular adhesion molecules(ICAM-1),matrix metal oproteinases 2(MMP-2)and MMP-9contents in cultured pulmonary microvascular endothelial cel s(PMVECs)in rats after perfluoroisobutylene(PFIB) exposure. METHODS PMVECs were separated and purified using a modified method of implantation of pulmonary tissues. After identification,PMVECs were divided into the normal control group and the PFIB-exposed groups(n=3). The PFIB-exposed groups inhaled PFIB at the concentration of 200 mg·m-3for 5 min in a flow-past header,while the normal control group were PMVECs in quiescent condition. The supernatants and lysates of PMVECs were harvested at 0.5,1,2,4 and 8 h,respectively,after execution. The contents of TNF-α,IL-1β,ICAM-1,MMP-2 and MMP-9 were measured by ELISA,and the activity of MMP-2 and MMP-9 was measured by gelatin zymography. RESULTS1 According to the morphologic characteristics of cell growth and the expression of specificity antigens and the bind experiment of phytohemagglutinin,the cells separated and purified by modified method shared the characteristics of PMVECs. 2 TNF-α was rapidly expressed by PMVECs at 0.5 h post PFIB stimulation and the maximum value was achieved at 2 h post PFIB stimulation(P<0.05). The newly synthesized TNF-α was slowly released out of the cells. The maximum TNF-α in the supernatant was achieved at 4 h post stimulation. 3 Within 2 h of stimulation,PMVECs synthesized a large amount of IL-1β and peaks at 2 h. However,IL-1β was never released to the extracellular milieu. 4 The amount of ICAM-1 was rapidly synthesized by PMVECs after PFIB stimulation,but at a low level. 5 After stimulation with PFIB,MMP-2 in the supernatant of PMVECs culture was gradually increased,peaked at 2 h and then decreased subsequently. The biological activity of MMP-2 in the supernatant was also enhanced after PFIB stimulation. PFIB did not stimulate synthesis or secretion of MMP-9,indicating that PMVECs were not the main source of MMP-9 during PFIB inhalation-induced acute lung injury.CONCLUSION PFIB stimulates the surviving PMVECs to synthesize a large amount of TNF-α,IL-1β,MMP-2 and conjunctive ICAM-1.更多还原