肝素抑制脂多糖诱导单核细胞与内皮细胞的黏附作用
Heparin inhibits lipopolysaccharide-induced adhesion of monocytes to endothelial cells
目的 探讨肝素对脂多糖(LPS)刺激人脐静脉内皮细胞(HUVEC)分泌单核细胞趋化蛋白1 (MCP-1)水平以及单核细胞与内皮细胞黏附作用的影响.方法 对HUVEC进行体外培养,取第4~5代用于本次实验.将细胞分成磷酸盐缓冲液(PBS)对照组、肝素对照组、LPS组、肝素+ LPS组.LPS组加入10 mg/L的LPS ;PBS对照组则加入等量PBS ;肝素对照组则加入10 kU/L普通肝素;肝素+ LPS组在进行LPS 刺激前15 min加入10 kU/L的普通肝素.各组于LPS刺激后6 h、12 h收集细胞,通过实时荧光定量反转录-聚合酶链反应(qRT-PCR)测定细胞中MCP-1 mRNA表达;各组预处理后加入荧光染料标记的人单核细胞株THP-1共同避光孵育1 h,在荧光显微镜下观察THP-1与HUVEC的黏附密度.结果 与PBS对照组相比,LPS刺激后6 h和12 h细胞MCP-1 mRNA表达明显增加,6 h达到高峰,然后逐渐降低,但12 h仍明显高于PBS对照组〔2-ΔΔCt:6 h为16.41(15.03,18.00)比1.00(0.80,1.26),12 h为9.27(8.11,9.85)比1.00(0.84,1.20),均P<0.05〕.用肝素预处理可使LPS诱导的MCP-1 mRNA表达明显降低〔2-ΔΔCt :6 h为2.06(1.72,2.46)比16.41(15.03,18.00), 12 h为2.46(2.19,4.56)比9.27(8.11,9.85),均P<0.05〕.肝素对照组MCP-1 mRNA表达与PBS对照组相比差异无统计学意义〔2-ΔΔCt:6 h为1.47(1.29,1.65)比1.00(0.80,1.26),12 h为2.69(2.58,2.77)比1.00(0.84,1.20),均P>0.05〕.荧光显微镜下观察显示,LPS刺激可促进THP-1与HUVEC的黏附;肝素预处理可抑制LPS刺激的THP-1与HUVEC黏附.结论 肝素预处理可以抑制MCP-1的表达,从而减少THP-1与HUVEC的黏附,在脓毒症中发挥保护作用.
更多Objective To investigate the effects of heparin on the secretion of monocyte chemotactic protein-1 (MCP-1) in human umbilical vein endothelial cells (HUVEC) and the adhesion of monocytes to endothelial cells stimulated by lipopolysaccharide (LPS). Methods HUVEC were cultured in vitro, and the cells between generation 4 and 5 were used for the experiments. The cells were divided into phosphate buffer saline (PBS) control group, heparin control group, LPS group, and heparin+LPS group. The LPS group was challenged with LPS 10 mg/L; the PBS control group was added with the same amount of PBS; the heparin group was added with 10 kU/L unfractionated heparin; the heparin+LPS group was treated with 10 kU/L unfractionated heparin 15 minutes before LPS stimulation. The cells were harvested at 6 hours and 12 hours after LPS stimulation in each group, and the MCP-1 mRNA expression was determined by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (qRT-PCR). After incubation with each group, the fluorescent dyelabeled human monocyte cell line THP-1 was cultured with each group for 1 hour in the dark, and the adhesion density of THP-1 and HUVEC was observed under fluorescence microscope. Results Compared with the PBS control group, the MCP-1 mRNA expression significantly increased at 6 hours and 12 hours after LPS stimulation and peaked at 6 hours, then decreased gradually, but remained significantly higher than the PBS control group at 12 hours [2-ΔΔCt: 16.41 (15.03, 18.00) vs. 1.00 (0.80, 1.26) at 6 hours, 9.27 (8.11, 9.85) vs. 1.00 (0.84, 1.20) at 12 hours, both P < 0.05]. Heparin preconditioning significantly reduced LPS-induced MCP-1 mRNA expression [2-ΔΔCt: 2.06 (1.72, 2.46) vs. 16.41 (15.03, 18.00) at 6 hours, 2.46 (2.19, 4.56) vs. 9.27 (8.11, 9.85) at 12 hours, both P < 0.05]. There was no significant difference in MCP-1 mRNA expression between the heparin control group and the PBS control group [2-ΔΔCt: 1.47 (1.29, 1.65) vs. 1.00 (0.80, 1.26) at 6 hours, 2.69 (2.58, 2.77) vs. 1.00 (0.84, 1.20) at 12 hours, both P > 0.05]. Fluorescence microscopy observation showed that LPS stimulation could promote the adhesion of THP-1 to HUVEC; heparin preconditioning could inhibit the adhesion of THP-1 to HUVEC stimulated by LPS. Conclusion Heparin preconditioning could inhibit the MCP-1 mRNA expression , thereby reduce the adhesion of THP-1 to HUVEC, thus play a protective role in sepsis.
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