过表达线粒体融合蛋白2对阿霉素诱导下血管内皮祖细胞衰老及凋亡的影响  

Overexpression of mitofusin2 inhibits senescence and apoptosis of endothelial progenitor cells induced by doxorubicin

作　　者：高帅 泮思林[1] 罗刚[1] 纪志娴[1] 孙宏晓 Gao Shuai;Pan Silin;Luo Gang;Ji Zhixian;Sun Hongxiao(Heart Center,Qingdao Women and Children’s Hospital,Qingdao University,Qingdao 266034,China)

机构地区：[1]青岛大学附属妇女儿童医院,青岛市妇女儿童医院心脏中心,266034

出　　处：《中华实验外科杂志》2021年第5期810-813,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81770316、81970249);泰山学者工程资助(2018)。

摘　　要：目的探讨过表达线粒体融合蛋白2(Mfn2)对阿霉素(Dox)诱导下血管内皮祖细胞(EPCs)衰老及凋亡的影响。方法抽取SD雄性大鼠外周动脉血,分离培养EPCs,进一步分为,正常EPCs组、空载体组、Mfn2过表达组,经0.2μmol/L和2.0μmol/L Dox诱导,通过激光共聚焦显微镜观察线粒体分裂情况;通过β-半乳糖苷酶活性检测、原位末端标记法(TUNEL)染色,检测细胞衰老和凋亡情况;蛋白印迹法检测细胞衰老标志蛋白多肿瘤抑制基因1(p16INK4A)的表达;实时荧光定量聚合酶链反应检测内皮型一氧化氮合酶(eNOS)及血管性血友病因子(vWF)的表达。多组间均数比较采用单因素方差分析。结果在0.2μmol/L和2.0μmol/L Dox处理下,Mfn2过表达组[(5.83±1.71)%、(6.72±0.57)%]线粒体分裂的细胞比例低于正常EPCs组[(19.16±0.82)%、(68.56±5.31)%]和空载体组[(17.53±2.86)%、(66.54±2.86)%,F=40.403、302.725,P<0.05],差异均有统计学意义;在0.2μmol/L Dox处理下,Mfn2过表达组[(15.0±0.33)%]β-半乳糖苷酶活性升高的细胞比例低于正常EPCs组[(35.0±0.61)%]和空载体组[(36.0±1.23)%,F=637.291,P<0.05],差异均有统计学意义;在2μmol/L Dox处理下,Mfn2过表达组[(11.90±0.50)%]TUNEL+细胞的比例低于正常EPCs组[(34.50±0.80)%]和空载体组[(35.20±1.60)%,F=458.327,P<0.05],差异均有统计学意义;在0.2μmol/L Dox处理条件下,Mfn2过表达组eNOS mRNA(0.65±0.02)和vWF mRNA(0.65±0.01)的相对表达高于正常EPCs组(0.43±0.03、0.50±0.02)以及空载体组(0.34±0.02、0.48±0.02,F=134.643、71.646,P<0.05),差异均有统计学意义;在2.0μmol/L Dox处理条件下,Mfn2过表达组eNOS mRNA(0.71±0.02)和vWF mRNA(0.66±0.03)的表达高于正常EPCs组(0.33±0.02、0.28±0.02)以及空载体组(0.38±0.03、0.35±0.01,F=225.712、262.934,P<0.05),差异均有统计学意义。结论过表达Mfn2可以抑制线粒体分裂,拮抗Dox诱导下EPCs的衰老及凋亡,恢复EPCs的功能。Objective To investigate the function of mitofusin2(Mfn2)on senescence and apoptosis of endothelial progenitor cells(EPCs)induced by doxorubicin(Dox).Methods EPCs isolated from peripheral blood of rats were divided into three groups,control group,mock-vehicle group and Mfn2 overexpression group.EPCs were treated with 0.2μmol/L or 2.0μmol/L Dox.Mitochondrial fission results were confirmed by laser-scanning confocal microscopy,cellular senescence and apoptosis were examined byβgalactosidase(β-gal)activity assays and terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)staining.Western blotting and reverse transcription quantitative real-time polymerase chain reaction were applied to detect the expression of inhibitor of cyclin-dependent kinase 4a(P16INK4A).The measurement data in accordance with normal distribution were expressed as mean±standard deviation,and the means of multiple groups were compared by single factor analysis of variance.Results Compared with control group[(19.16±0.82)%,(68.56±5.31)%]and mock-vehicle group[(17.53±2.86)%,(66.54±2.86)%],the percentage of cells with mitochondrial fission was significantly decreased in Mfn2 group[(5.83±1.71)%,(6.72±0.57)%]with the treatment of 0.2μmol/L and 2.0μmol/L Dox(F=40.403,302.725,P<0.05).After treatment with 0.2μmol/L Dox,the percentage ofβ-gal+cells in Mfn2 group[(15.0±0.33)%]was significantly decreased(F=637.291,P<0.05)as compared with control group[(35.00±0.61)%]and mock-vehicle group[(36.00±1.23)%].Furthermore,after treatment with 2μmol/L Dox,the percentage of TUNEL+cells in Mfn2 group[(11.90±0.50)%]was significantly less than in control group[(34.50±0.80)%]and mock-vehicle group[(35.20±1.60)%](F=458.327,P<0.05).In the end,after treatment with 0.2μmol/L Dox,the mRNA levels of eNOS(0.65±0.02)and vWF(0.65±0.01)in Mfn2 group were significantly increased as compared with control group[(0.43±0.03,0.50±0.02)]and mock-vehicle group[(0.34±0.02,0.48±0.02),F=134.643,71.646,P<0.05].Meanwhile,under 2μmol/L Dox treatmen

关 键 词：线粒体融合蛋白2 内皮祖细胞 基因转染 阿霉素 线粒体分裂 

分 类 号：R965[医药卫生—药理学]