摘要
目的探讨过表达钠氢交换体1(NHE1)对RAW264.7细胞三磷酸腺苷结合盒转运体A1(ABCA1)蛋白表达的影响。方法采用Ad NHE1腺病毒感染RAW264.7细胞,Western blot法检测NHE1-EGFP融合蛋白的表达,激光共聚焦显微镜技术检测NHE1-EGFP融合蛋白细胞内定位,酸负载p H回复检测NHE1活性,Western blot法检测NHE1-EGFP融合蛋白对RAW264.7细胞ABCA1蛋白水平及钙蛋白酶(calpain)活性的影响,加入calpain抑制剂N-乙酰基-L-亮氨酰-L-亮氨酰-L-正亮氨酸(ALLN),Western blot法检测肝脏X受体激动剂TO-901317诱导ABCA1蛋白表达水平的影响。结果 Ad NHE1腺病毒感染RAW264.7细胞后,高表达NHE1-EGFP融合蛋白,定位在细胞质及细胞膜。NHE1-EGFP融合蛋白可降低ABCA1蛋白的表达水平并提高calpain活性,而calpain抑制剂ALLN能阻断ABCA1蛋白表达水平降低。结论 NHE1过表达通过上调calpain活性而降低ABCA1蛋白表达水平。
Objective To investigate the effect of over-expressed Na＋/H＋ exchanger 1（ NHE1） on the protein expression of adenosine three phosphate binding cassette transporter A1（ ABCA1） in RAW264.7 cells.Methods RAW264.7 cells were infected with the adenoviral vector encoding NHE1-EGFP（ Ad NHE1）.The infected RAW264.7 cells were subjected to Western blot analysis for NHE1-EGFP fusion protein.The subcellular localization of NHE1-EGFP fusion protein was observed by confocal laser scanning microscopy.NHE1 activity was measured by the method of p H recovery in response to an acute acid pulse.Furthermore,Western blotting was performed to determine ABCA1 protein levels and calpain activity in NHE1-overexpressing RAW264.7 cells.The effect of calpain inhibitor N-acetyl-L-leucyl-L-leucyl-L-norleucinal（ ALLN） on ABCA1 protein levels in the presence of TO-901317 was examined by Western blotting.Results NHE1-EGFP fusion protein was highly expressed and localized in cytoplasm and cell membrane of RAW264.7 cells infected with Ad NHE1.NHE1-EGFP fusion protein reduced ABCA1 protein expression and increased calpain activity.The calpain inhibitor ALLN blocked the decrease of ABCA1 protein expression.Conclusion Overexpressed NHE1 suppresses the expression of ABCA1 protein via increasing the calpain activity in RAW264.7 cells.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2017年第1期12-16,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(31260250)
