普鲁士蓝纳米粒对过氧化氢诱导脂肪间充质干细胞凋亡的影响  

Effect of Prussian blue nanoparticles on apoptosis of adipose mesenchymal stem cells induced by H_(2)O_(2)

作　　者：孙宝俊 赵风景[2] 章文锋[2] 王红霞[3] 白丽民 徐刚[4,5] Sun Baojun;Zhao Fengjing;Zhang Wenfeng;Wang Hongxia;Bai Limin;Xu Gang(Medical College of Yangzhou University,Yangzhou 225001,China;Department of Hand Surgery,Hangzhou Plastic Surgery Hospital,Hangzhou 310014,China;Department of Clinical Laboratory,Hangzhou Plastic Surgery Hospital,Hangzhou 310014,China;Department of Burns and Plastic Surgery,Northern Jiangsu People’s Hospital,Yangzhou 225001,China;Clinical College of Yangzhou University,Yangzhou 225001,China)

机构地区：[1]扬州大学医学院,扬州225001 [2]杭州整形医院手外科,杭州310014 [3]杭州整形医院检验科,杭州310014 [4]苏北人民医院烧伤整形科,扬州225001 [5]扬州大学临床医学院,扬州225001

出　　处：《中华实验外科杂志》2023年第12期2548-2553,共6页Chinese Journal of Experimental Surgery

基　　金：杭州市医药卫生科技项目(B20200041)。

摘　　要：目的探究普鲁士蓝纳米粒(PBNPs)对过氧化氢(H_(2)O_(2))诱导脂肪间充质干细胞(ADSCs)凋亡的影响及其相关机制。方法水热合成法制备PBNPs,电镜、傅里叶变换红外光谱等表征PBNPs合成;流式鉴定ADSCs表面标志物CD29、CD90、CD44、CD73、CD45、CD34;噻唑蓝(MTT)法检测H_(2)O_(2)、PBNPs细胞毒性;细胞分为Control组、H_(2)O_(2)组(200μmol/L H_(2)O_(2)处理24 h)、L-PBNPs组(10μg/ml PBNPs预处理12 h后,200μmol/L H_(2)O_(2)处理24 h)、H-PBNPs组(20μg/ml PBNPs预处理12 h后,200μmol/L H_(2)O_(2)处理24 h);活性氧探针检测ADSCs的活性氧水平;流式细胞仪分析ADSCs的凋亡水平;蛋白质印迹法检测凋亡相关蛋白表达水平;两组间比较采用t检验,多组间比较采用单因素方差分析(One-way ANOVA)。结果PBNPs为正立方体,表面Fe、C、N等元素均匀分布;ADSCs CD29、CD90、CD44、CD73阳性,CD45、CD34阴性;L-PBNPs组和H-PBNPs组活性氧平均荧光强度、凋亡率显著低于H_(2)O_(2)组[7.29±1.38、2.38±0.21比16.35±3.86,t=3.828、6.259,P<0.05;(10.21±2.27)%、(6.01±0.57)%比(25.42±3.10)%,t=6.857、10.67,P<0.05];与H_(2)O_(2)组比较,L-PBNPs组半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、细胞周期蛋白依赖性激酶抑制剂1A(p21)、B细胞淋巴瘤因子-2(bcl-2)相关X蛋白(bax)显著下调(1.877±0.076比3.087±0.179,t=6.21,P<0.05;1.887±0.118比2.627±0.052,t=5.72,P<0.05;1.950±0.196比3.133±0.291,t=3.37,P<0.05;3.567±0.398比5.681±0.455,t=3.49,P<0.05),bcl-2显著上调(0.363±0.064比0.131±0.025,t=3.38,P<0.05);与H_(2)O_(2)组比较,H-PBNPs组Caspase-9、Caspase-3、p21、bax显著下调(1.083±0.095比3.087±0.179,t=9.88,P<0.05;1.117±0.066比2.627±0.052,t=17.78,P<0.05;1.067±0.095比3.133±0.291,t=6.76,P<0.05;1.740±0.366比5.681±0.455,t=6.74,P<0.05),bcl-2显著上调(0.802±0.026比0.131±0.025,t=18.49,P<0.05)。结论PBNPs对氧化应激处理的ADSCs表现出良好的抗凋亡能力。Objective To investigate the effect of Prussian blue nanoparticles(PBNPs)on apoptosis of adipose mesenchymal stem cells(ADSCs)induced by H_(2)O_(2) and its related mechanism.Methods PBNPs were prepared by hydrothermal synthesis,and characterized by scanning electron microscopy(SEM),transmission electron microscopy(TEM),scanning transmission electron microscopy(STEM),fourier transform infrared spectroscopy(FTIR)and methods.ADSCs were extracted by collagenase digestion,and identified by CD29,CD90,CD44,CD73,CD45,CD34;the cytotoxicity of H_(2)O_(2) and PBNPs was detected by 3-[4,5-diamethylthiazol-2-yl]2,5-diphenyltetrazoliumbromide(MTT)assay;the cells were divided into Control group,H_(2)O_(2) group(200μmol/L H_(2)O_(2) for 24 h),L-PBNPs group(10μg/ml PBNPs for 12 h,then 200μmol/L H_(2)O_(2) for 24 h)and H-PBNPs group(20μg/ml PBNPs for 12 h,then 200μmol/L H_(2)O_(2) for 24 h);reactive oxygen species(ROS)levels were detected by ROS probe;the apoptosis rates were analyzed by flow cytometry;the protein of apoptosis related were detected by Western blotting.The comparison between two groups was performed by t test,and the comparison between multiple groups was performed by one-way analysis of variance(One-way ANOVA).Results SEM,TEM and STEM results showed that PBNPs owned a cube structure,Fe,C,N and other elements were evenly distributed on the surface.ADSCs were positive for CD29,CD90,CD44 and CD73,negative for CD45 and CD34.The mean fluorescence intensity of ROS,apoptosis rates of L-PBNPs group and H-PBNPs group were significantly lower than those of H_(2)O_(2) group[7.29±1.38,2.38±0.21 vs.16.35±3.86,t=3.828,6.259,P<0.05;(10.21±2.27)%,(6.01±0.57)%vs.(25.42±3.10)%,t=6.857,10.67,P<0.05],the difference was statistically significant.The cysteine-requiring aspartate protease-9(Caspase-9),cysteine-requiring aspartate protease-3(Caspase-3),cyclin dependent kinase inhibitor 1A(p21)and B cell lymphoma-2 associated X(bax)level of L-PBNPs group were significantly lower than those of H_(2)O_(2) group(1.877±0.076 vs.3.

关 键 词：普鲁士蓝纳米粒 脂肪间充质干细胞 活性氧 凋亡 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]