低浓度纳米银促进脐带间充质干细胞成骨分化的研究  

Silver nanoparticles promote osteogenic differentiation of human umbilical cord mesenchymal stem cells at non-cytotoxic concentration

作　　者：毕懿康 黄默冉 周路 吴琪 时潇 耿彩云 李红艳 王海妹 张艺 亓建洪 秦晖[2] BI Yikang;HUANG Moran;ZHOU Lu;WU Qi;SHI Xiao;GENG Caiyun;LI Hongyan;WANG Haimei;ZHANG Yi;QI Jianhong;QIN Hui(Institute of Sports Medicine, Shandong First Medical University, Tai’an 271016,China;Department of Orthopedics, the Sixth People’s Hospital Affilated to Shanghai Jiaotong University, Shanghai 200233,China;Department of Rehabilitation ,Tai’an Maternal and Child Health Hospital, Tai’an 271016,China)

机构地区：[1]山东第一医科大学运动医学研究所,泰安271016 [2]上海交通大学附属第六人民医院骨科,200233 [3]泰安市妇幼保健院康复科,271016

出　　处：《国际骨科学杂志》2019年第3期178-186,共9页International Journal of Orthopaedics

基　　金：山东省自然科学基金(ZR2017LH018B)

摘　　要：目的研究纳米银对人脐带间充质干细胞(hUCMSC)的毒性作用及成骨分化影响。方法首先,从人脐带中分离出hUCMSC,通过免疫荧光双染对hUCMSC进行细胞鉴定。将纳米银及硝酸银作用于hUCMSC24 h,用CCK-8细胞活力测试检测它们的细胞毒性,得出最大安全质量浓度。以此质量浓度的纳米银及硝酸银作用于hUCMSC,以碱性磷酸酶(ALP)活力、茜素红染色及定量逆转录聚合酶链反应(qRT-PCR)检测纳米银及硝酸银对hUCMSC成骨分化的影响。用罗丹明-鬼笔环肽染色检测纳米银对hUCMSC细胞骨架的作用,用蛋白免疫印迹法测试纳米银对hUCMSC RhoA蛋白水平的影响。结果纳米银及硝酸银的细胞毒性呈剂量依赖性,在纳米银4μg/mL及硝酸银2μg/mL时,无细胞毒性作用发生。与空白组及硝酸银组相比,在4μg/mL纳米银作用下,hUCMSC的ALP活性更高,形成钙结节的量更多,且成骨分化相关基因(Runt相关转录因子2、骨桥蛋白、骨钙蛋白、骨形态发生蛋白-2、Ⅰ型胶原蛋白α1链)的表达更高。罗丹明-鬼笔环肽染色显示,纳米银可诱发hUCMSC的肌动蛋白聚合,增加细胞骨架张力。蛋白免疫印迹法测试显示,纳米银提高了活化RhoA蛋白水平,而硝酸银无此作用。结论纳米银可在合适的质量浓度通过活化RhoA蛋白、诱导肌动蛋白聚合及增加细胞骨架张力提高hUCMSC的成骨分化能力,且此作用与银离子无关。Objective To investigate the cytotoxicity and osteogenic differentiation of human umbilical cord mesenchymal stem cells(hUCMSC)induced by silver nanoparticles(Ag-NPs).Methods Initially,hUCMSC was isolated from human umbilical cord and identified by double immunofluorescence staining.Ag-NPs or AgNO3 were exposed to hUCMSC for 24 hours.Cytotoxicity was measured by the Cell Counting Kit-8(CCK-8)test.The effect of Ag-NPs or AgNO3 at the maximum safety concentration by the CCK-8(CCK-8)test on osteogenic differentiation of hUCMSC was assessed by alkaline phosphatase activity,Alizarin red staining,and the quantitative reverse transcription polymerase chain reaction.Lastly,the effect of Ag-NPs or AgNO3 on hUCMSC actin cytoskeleton organization and RhoA activity was assessed by rhodamine-phalloidin staining and Western blotting.Results Compared with the control and AgNO3 group,the alkaline phosphatase activity and calcium nodule formation of hUCMSC were higher under the action of 4μg/mL Ag-NPs,and the expression of osteogenic differentiation related genes including core binding protein factor 2,typeⅠcollagen,osteocalcin,osteopontin,and bone morphogenetic protein2,was higher,indicating that Ag-NPs promoted osteogenic differentiation of hUCMSC.Rhodamine-phalloidin staining showed that Ag-NPs induced actin aggregation and increased cytoskeleton tension.Western blotting showed that Ag-NPs increased the level of activated RhoA protein,while AgNO3 did not.Conclusion Ag-NPs can promote osteogenic differentiation of hUCMSC at a suitable concentration,independently of Ag+ions,thus being suitable to incorporate into tissue-engineered scaffolds that utilize hUCMSC as seed cells.

关 键 词：纳米银 成骨分化 干细胞 细胞毒性 RHOA蛋白 

分 类 号：R68[医药卫生—骨科学]