不同时程低氧暴露对大鼠髓源性细胞HIF-1α与hGlyrichin表达影响的研究

Investigation of different hypoxia exposure effect on the HIF-1α and hGlyrichin expression in rat's myeloid-derived cells

目的:通过不同时程间歇性低氧暴露后大鼠髓源性细胞指标测试,观察分析间歇性低氧对低氧诱导因子HIF-1α与人源性抗菌肽h Glyrichin表达变化趋势的影响,探讨低氧与机体固有免疫机能变化之间的可能联系。方法:雄性Wistar大鼠60只随机分为对照组、间歇性低氧暴露12、24、36、48、72小时组,低氧后尾静脉取静脉血,用淋巴细胞分离液分离收集多形核白细胞(polymorphonuclear leukocyte,PMN),以Trizol提取RNA用于进行RT-PCR,同时以RIPA细胞裂解液制备细胞蛋白样品,以western blot检测HIF-1α表达变化,并通过双向电泳分析间歇性低氧暴露72小时后蛋白质表达差异。结果:大鼠多形核白细胞HIF-1αmRNA水平在间歇性低氧暴露累计36小时后显著升高,且随时程延长其表达水平进一步显著上升;HIF-1α蛋白表达水平在间歇性低氧暴露累计24小时后即开始显著增加,且随时程延长有进一步升高趋势;在间歇性低氧暴露72小时后蛋白差异表达分析只鉴定出一种表达显著上调的蛋白质tropomyosin 4,其与低氧诱导通路无直接联系;富含甘氨酸的抗菌肽h Glyrichin表达水平在间歇性低氧暴露后有上升趋势,但不同时程时间点之间未见有显著差异,与HIF-1α表达水平在间歇性低氧暴露36小时后即显著上调趋势并不一致。结论:在间歇性低氧暴露一定时程后HIF-1αmRNA水平与蛋白表达水平均有显著上升,但未能证实髓源性细胞中HIF-1α表达水平升高可能上调抗菌肽h Glyrichin表达的假设。

Objective：In order to provide a new insight upon the interactions between hypoxia and in-nate immunity responses,we analyzed the impact of on hypoxia-inducible factor 1 and a novel hu-man origined antimicrobial peptides hGlyrichin′s expression in rat′s myeloid-derived polymorphonu-clear leukocytes during different intermittent hypoxia exposure time course. Methods：60 Wistar male rats were randomly divided into 6 grounps as control and intermittent hypoxia for 12 ,24 ,36 ,48,72 hours,intermittent hypoxic was taken with hypoxico system which mimic the 2500 meters al-titude. Vein blood samples were collected and then polymorphonuclear leukocyte were separated by lymphocyte separation medium . RNA was extracted with Trizol for RT-PCR,while protein sample was prepared by RIPA lysis buffer for western blot to detect the expression of HIF-1α. Protein dif-ferential express pattern was determined by two -dimensional electrophoresis and mass spectrum. Results：HIF-1α mRNA level was significantly increased at intermittent hypoxia accumulated for 36 hours,and the expression level further increased significantly with time prolonged;HIF-1αprotein levels increased significant at intermittent hypoxia totaled for 24 hours and with a further increasing trend during time prolonged. Protein differentially expression only identified a protein tropomyosin 4,it showed no direct relationship with the hypoxia-inducible pathway. hGlyrichin expression only had an upward trend even after intermittent hypoxic for 72 hours,no significant difference was found consistent with HIF-1α expression changed. Conclusions：We found a significant increase in both the HIF-1αmRNA level and protein expression level during the time course by intermittent hypoxia stress,but failed to confirm the possible assume that the antimicrobial peptide expression level would be upregulated by the increased HIF-1α level in myeloid-derived cells.