摘要
目的:探讨CD137信号是否通过调控有氧糖酵解途径促进小鼠肺动脉内皮细胞(PAECs)增殖。方法:将小鼠PAECs分组如下:(1)空白对照组、同型对照组、刺激因子TNF-α(10μg/L)组、ET-1(10 mmol/L)和5-HT(1μmol/L)组,各组刺激细胞24 h;(2)选择TNF-α刺激细胞24 h后分为对照组、CD137激动剂组(CD137L重组蛋白5 mg/L,激动CD137-CD137L信号)、c-Myc抑制剂组(c-Myc抑制剂10074-G5 10μmol/L,DMSO溶解,预处理细胞30 min,再加入CD137L重组蛋白)和DMSO组(加入与c-Myc抑制剂组等量DMSO预处理细胞30 min,再予CD137L重组蛋白刺激);(3)TNF-α刺激细胞24 h后分为对照组、CD137激动剂组和2-脱氧葡萄糖(2-DG)组(10 mmol/L糖酵解抑制剂2-DG预处理细胞30 min,再加入CD137L重组蛋白)。分别采用流式细胞术及Western blot检测细胞CD137膜蛋白及总蛋白表达。Western blot及酶学分析分别检测细胞己糖激酶(HK2)、6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(PFKFB3)和c-Myc蛋白表达及酶活性。葡萄糖氧化酶法及乳酸脱氢酶比色法检测细胞葡萄糖摄取量及乳酸水平。CCK-8法和EdU法检测细胞增殖。结果:与对照组相比,刺激因子TNF-α、ET-1及5-HT刺激24 h可明显增加PAECs的CD137膜蛋白及总蛋白的表达(P<0.05)。CD137激动剂组细胞HK2及PFKFB3蛋白表达及酶活性显著高于对照组;CD137激动剂组细胞乳酸水平高于对照组,葡萄糖消耗较对照组多(P<0.05)。激活CD137信号后PAECs的c-Myc蛋白表达高于对照组,而应用c-Myc抑制剂10074-G5可显著抑制CD137信号的促糖酵解作用(P<0.05)。CCK-8及EdU增殖试验显示激活CD137信号可显著促进PAECs增殖,有氧糖酵解抑制剂2-DG可抑制其促细胞增殖作用(P<0.05)。结论:CD137信号可能通过上调c-Myc增强小鼠PAECs有氧糖酵解,进而促进细胞增殖。
AIM: To investigate whether CD137 signaling molecules promote the proliferation of pulmonary artery endothelial cells(PAECs) by aerobic glycolysis. METHODS: The experiments of mouse PAECs were performed as follows.(1) Stimulating factors TNF-α(10 μg/L), ET-1(10 mmol/L) and 5-HT(1 μmol/L) were used to stimulate the cells for 24 h.(2) After stimulation with TNF-α for 24 h, the cells were divided into control group, CD137 agonist group(treatment with 5 mg/L CD137 L recombinant protein to activate CD137-CD137 L signaling), c-Myc inhibitor group(pretreatment with 10 μmol/L c-Myc inhibitor 10074-G5, dissolved in DMSO, for 30 min, followed by treatment with 5 mg/L CD137 L recombinant protein) and DMSO group(pretreated with DMSO at the same volume to c-Myc inhibitor group for 30 min followed by CD137 L recombinant protein treatment).(3) After stimulated with TNF-α for 24 h, the cells were divided into control group, CD137 agonist group and 2-deoxyglucose(2-DG) group(pretreatment with 10 mmol/L glycolysis inhibitor 2-DG for 30 min followed by CD137 L recombinant protein treatment).The expression of membrane protein and total protein of CD137 in the PAECs was detected by flow cytometry and Western blot, respectively. The protein levels of glycolytic enzymes such as hexokinase(HK2), 6-phosphofructo-2-kinase/fructose-2,6-diphosphatase 3(PFKFB3) and c-Myc were measured by Western blot. The enzyme activity of HK2 and PFKFB3 was detected by HK2 kit and PFK kit, respectively. Glucose oxidase method was used to measure the glucose uptake rate, and lactate colorimetric assay was conducted for analyzing lactic acid production. CCK-8 assay and EdU staining were used to detect proliferation of the PAECs. RESULTS: Compared with control group, TNF-α, ET-1 and 5-HT significantly increased the expression of CD137 membrane protein and total protein in the PAECs(P<0.05). The protein levels and enzyme activity of HK2 and PFKFB3 protein in CD137 agonist group were significantly higher than those in control group(P<0.05). Compared with
作者
蒋美萍
陈蕊
刘培晶
严金川
JIANG Mei-ping;CHEN Rui;LIU Pei-jing;YAN Jin-chuan(Department of Cardiology, The Affiliated Hospital of Jiangsu University, Zhenjiang 212001,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2019年第6期968-974,共7页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81670405
No.81400208)
江苏省自然基金资助项目(No.BK20161355
BK20181227)
镇江市心血管病临床医学研究中心资助项目(No.SS2018008)
镇江市社会发展基金资助项目(No.SH2018043)
