地塞米松刺激THP-1细胞释放具有促凝血功能的组织因子阳性膜微粒

发布时间：2018-08-27 06:31

【摘要】：研究目的：探究激素刺激下,单核细胞所释放的膜微粒和组织因子(TF)阳性膜微粒的数量、促凝血功能。组织因子的表达和作用,以及激素对单核细胞内组织因子表达的影响。内毒素对单核细胞的作用是否能被激素促进或者抑制。研究方法：选用人单核细胞肿瘤细胞系THP-1为研究对象。用地塞米松0.1mM、0.01mM、0.001mM三种不同浓度分别刺激4h、12h、24h后,用流式细胞仪检测上清液中组织因子阳性膜微粒和总膜微粒的数量,以及TF阳性膜微粒所占比例。用一期法凝血时间测定细胞上清液的促凝血活性。用CD14抗体捕获膜微粒,测定膜微粒相关的组织因子活性。用荧光实时定量PCR测定细胞中组织因子的mRNA水平。研究结果：除4h-0.001mM组外,激素刺激组释放的TF阳性膜微粒数量比基础值高出1.5-3倍,总膜微粒高出基础值1.3-2.5倍。地塞米松的剂量与TF阳性膜微粒数量正相关(r=0.673,p0.001),与总膜微粒数量正相关(r=0.804,p=0.001)。激素对LPS刺激THP-1细胞释放TF阳性膜微粒没有促进作用。地塞米松刺激THP-1细胞后释放的TF阳性膜微粒表面的TF具有活性。地塞米松刺激THP-1细胞的上清液的凝血时间比阴性对照缩短(p0.05),4h组和24h组的凝血时间与同一样本的上清液中TF阳性膜微粒数量负相关(4h r=-0.762p=0.028；12h r=-0.833p=0.010).地塞米松刺激4h时显著减少了TF-mRNA的水平(p0.01),0.01mM浓度在12h和24h刺激时间能增加TF-mRNA (p0.01)。TF-mRNA水平与凝血时间没有相关性。结论：地塞米松刺激THP-1细胞释放高于基础量的TF阳性膜微粒,这些膜微粒具有TF活性,其数量与地塞米松剂量正相关,与促凝血作用强度存在一定的相关性。地塞米松的这种作用不是来源于转录水平的调控。
[Abstract]:Aim: to investigate the number of membrane particles and tissue factor (TF) positive membrane particles released by monocytes stimulated by hormones, and to investigate the coagulation function. The expression and function of tissue factor and the effect of hormone on the expression of tissue factor in monocytes. Whether the action of endotoxin on monocytes can be promoted or inhibited by hormones. Methods: human monocyte tumor cell line THP-1 was selected as the research object. Three different concentrations of dexamethasone 0.1 mMN 0.01mMN 0.001mm were used to stimulate 4 h and 12 h respectively for 24 h. Flow cytometry was used to detect the number of tissue factor positive membrane particles and the total membrane particles in the supernatant and the proportion of TF positive membrane particles in the supernatant. The procoagulant activity of supernatant was measured by one-stage coagulation time. CD14 antibody was used to capture the membrane particles and the tissue factor activity related to the membrane particles was determined. The mRNA level of tissue factor in cells was measured by fluorescence real-time quantitative PCR. Results: except for 4h-0.001mM group, the number of TF positive membrane particles released by hormone stimulation group was 1.5-3 times higher than the base value, and the total membrane particles was 1.3-2.5 times higher than the base value. The dose of dexamethasone was positively correlated with the number of TF positive membrane particles (r = 0.673, p0.001), and with the number of total membrane particles (r = 0.804, p0. 001). Hormones did not promote the release of TF positive membrane particles from THP-1 cells stimulated by LPS. TF on the surface of TF positive membrane particles released by dexamethasone stimulated THP-1 cells was active. The coagulation time of the supernatant stimulated by dexamethasone in THP-1 cells was significantly shorter than that in the negative control group (p0.05) and the coagulation time was negatively correlated with the number of TF positive membrane particles in the supernatant of the same sample (4h r-0.762pt0. 028 h r=-0.833p=0.010). Dexamethasone significantly decreased the level of TF-mRNA (p0.01) 0.01 mm at 12 h and 24 h after stimulation, and increased the level of TF-mRNA (p0.01). TF-mRNA was not correlated with coagulation time. Conclusion: Dexamethasone stimulated THP-1 cells to release more TF positive membrane particles than the basic amount. These membrane particles have TF activity. The number of these particles is positively correlated with the dose of dexamethasone and has a certain correlation with the blood coagulation intensity. This effect of dexamethasone is not derived from the regulation of transcriptional levels.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R363

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