摘要
目的通过检测ASK1和PRKCD在单核细胞分化过程中的表达变化,探索其在门静脉高压症(PH)脾亢脾脏巨噬细胞(MΦ)功能变化中所起的作用。方法采用佛波酯诱导U937分化成为巨噬细胞样细胞,q-PCR检测不同分化阶段ASK1和PRKCD m RNA的表达,Western Blot检测诱导前后ASK1和PRKCD蛋白表达水平变化,ELISA法检测细胞分化过程中与吞噬功能相关的细胞因子IL-10和TNF-α的分泌情况,鸡红细胞吞噬试验鉴定诱导后细胞功能。结果随着PMA诱导U937细胞时间延长,ASK1和PRKCD基因表达水平下降,TNF-α的分泌量逐渐增加,而IL-10在诱导后24 h达到最大分泌量,随后又呈下降趋势;Western Blot结果显示,ASK1及p-ASK1蛋白表达水平较诱导前均显著上升,PRKCD及p-PRKCD蛋白表达水平显著下降;吞噬实验结果显示,诱导后的细胞具有一定的吞噬功能。结论在PMA诱导的U937细胞向单核细胞分化过程中,ASK1蛋白表达上调,PRKCD蛋白表达下调,与脾亢脾Mφ一致,并导致Mφ活性增强。
Objective To investigate the expression of ASK1 and PRKCD in the process of monocyte differentiation, and explore their role in functional changes of hypersplenism spleen macrophages（MΦ） in portal hypertension（PH）. Methods U937 cells were stimulated to differentiate into monocyte/macrophage- like cells by cultivation in PMA and the m RNA expressions of ASK1 and PRKCD were detected by q-PCR and the changes of protein expression were identified by western blot analysis. The secretion of phagocytose related cytokines such as IL-10 and TNF-a were tested by ELISA, and the function of the macrophagelike cells were studied by chicken red blood cell phagocytose test. Results The expressions of PRKCD and ASK1 m RNA were gradually decreased along with the cell differentiation, while the secretion of TNF-α was increased, IL-10 secretion reached a maximum at 24 h after PAM stimulation, and then gradually fell. The expression of ASK1 and p-ASK1 were rapidly increased compared with the non- stimulated U937 cells, while the expression of PRKCD and p- PRKCD were sightly declined. The phagocytose test show that U937 cells induced with PMA were able to swallow the chicken red blood cell.Conclusion Upregulated protein expression of ASK1 and p- ASK1 and down- regulated protein expression of PRKCD and p- PRKCD in the process of PMA induced monocyte differentiation, are consist with the expression changes of splenic macrophage phagocytosis in hypersplenism, which leads to increased activity of MΦ.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2015年第1期17-22,共6页
Journal of Southern Medical University
基金
国家自然科学基金(30901454)
福建省卫生厅青年科研课题计划资助项目(2009-2-68)~~
