双特异性磷酸酶5过表达/干扰RAW264.7细胞株建立及其对自噬调控的影响  

Establishment of Dual-Specific Phosphatase Overexpressed/Interfered RAW264.7 Cell Strain and Its Effect on Autophagy

作　　者：骆佳 曾瑾 杨易 王玉炯 Luo Jia;Zeng Jin;Yang Yi;Wang Yujiong(Key Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China,Ningxia University,Yinchuan 750021,China)

机构地区：[1]宁夏大学,西部特色生物资源保护与利用教育部重点实验室,银川750021

出　　处：《中国细胞生物学学报》2018年第8期1264-1274,共11页Chinese Journal of Cell Biology

基　　金：国家自然科学基金项目(批准号:31572494)资助的课题~~

摘　　要：为探究双特异性磷酸酶5(dual-specificity phosphatase 5,DUSP5)在巨噬细胞RAW264.7自噬中的调控作用,该研究采用慢病毒转染的技术方法建立DUSP5过表达/干扰RAW264.7细胞株,并用自噬激活剂雷帕霉素(rapamycin,Rapa)和抑制剂巴弗洛霉素A1(bafilomycin A1,Baf A1)分别对其进行刺激,使用蛋白免疫印迹技术(Western blot)、荧光定量PCR(Real-time fluorescence quantitative PCR,q RT-PCR)、单丹磺酰尸胺(monodansylcadaverine,MDC)染色以及免疫荧光等方法探究过表达或抑制DUSP5后对巨噬细胞RAW264.7自噬的影响。结果显示:DUSP5过表达/干扰慢病毒转染RAW264.7可显著提高/抑制DUSP5 m RNA和蛋白的表达量(P<0.01);Rapa刺激后,过表达DUSP5抑制自噬相关蛋白Beclin1和LC3II表达且自噬体形成减少,而抑制DUSP5表达结果与此相反;用Baf A1阻断DUSP5干扰RAW264.7稳定转染细胞株自噬流,DUSP5干扰RAW264.7稳定转染细胞株中LC3II表达量和自噬体数量均显著上调(P<0.01)。由此说明,DUSP5参与调控巨噬细胞自噬,可能通过抑制自噬体合成来阻碍巨噬细胞自噬进程。此结论为进一步探究巨噬细胞自噬调控机制提供了新的研究思路。To study the effect of dual-specificity phosphatase 5（DUSP5） on the autophagy of macrophages, they established DUSP5 overexpressed/interfered RAW264.7 cell strain by transfecting lentivirus vector. Besides, rapamycin（Rapa） and bafilomycin A1（Baf A1） were used to stimulate the cell strains. Western blot, realtime fluorescence quantitative PCR（q RT-PCR）, Monodansylcadaverine（MDC） staining and immunofluorescence were all applied to this research. The results showed that RAW264.7 transfected with DUSP5 overexpression/interference lentivirus could significantly increase/inhibit（P0.01） the expression of DUSP5 m RNA and protein.Overexpressing DUSP5 inhibited the expression of autophagy-related proteins Beclin1 and LC3 II and the formation of intracellular autophagosomes after Rapa stimulation, however, suppressing DUSP5 expression had reversed result. The expression of LC3 II and the number of autophagosomes were significantly increased after blocking autophagy flux of DUSP5 interfered RAW264.7 cell strain with Baf A1. In a conclusion, DUSP5 plays a role in the regulation of macrophages autophagy, it may inhibit autophagy by interrupting autophagosomes formation. This finding will provide a new idea for further exploring the regulation mechanism of macrophage autophagy.

关 键 词：双特异性磷酸酶5 巨噬细胞 自噬 

分 类 号：Q25[生物学—细胞生物学]