3D培养人脐带间充质干细胞来源的外泌体对成骨细胞分化的作用  

Effects of exosomes derived from 3D-cultured human umbilical cord mesenchymal stem cells on osteoblast differentiation

作　　者：陈瑞婧 冯韬锦 程实[4] 李上 于海宽 陈铭 李毅[2,3] 尹鹏滨 张里程 唐佩福[2,3] Chen Rui-Jing;Feng Tao-Jin;Cheng Shi;Li Shang;Yu Hai-Kuan;Chen Ming;Li Yi;Yin Peng-Bin;Zhang Li-Cheng;Tang Pei-Fu(Medical School of Chinese PLA,Beijing 100853,China;Department of Orthopedics,the Fourth Medical Center of Chinese PLA General Hospital,Beijing 100048,China;National Orthopedics and Sports Rehabilitation Clinical Research Center,Beijing 100048,China;Department of Orthopedics,the Second Affiliated Hospital of Harbin Medical University,Harbin,Heilongjiang 150086,China)

机构地区：[1]解放军医学院,北京100853 [2]解放军总医院第四医学中心骨科医学部,北京100048 [3]国家骨科与运动康复临床医学研究中心,北京100048 [4]哈尔滨医科大学附属第二医院骨科,黑龙江哈尔滨150086

出　　处：《解放军医学杂志》2023年第4期411-419,共9页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81972115,82002330)。

摘　　要：目的比较三维立体(3D)培养与传统贴壁培养(2D)人脐带间充质干细胞(hUC-MSCs)来源的外泌体促进成骨细胞分化能力的差异,探索前者在促进骨形成相关治疗中应用的可能性。方法将hUC-MSCs分为2D组与3D组分别进行培养,在普通光学显微镜下观察细胞的形态特征,同时应用钙黄绿素-AM/PI活死细胞双染法检测3D组细胞的活性;通过转录组测序筛选两组差异表达基因并进行GO富集分析。提取2D组与3D组细胞分泌的外泌体并分为2D外泌体(2D-Exo)组与3D外泌体(3D-Exo)组,通过透射电子显微镜、纳米颗粒跟踪分析及Western blotting对外泌体进行表征。在体外应用2D-Exo及3D-Exo干预乳鼠颅骨成骨细胞并进行成骨诱导分化,通过茜素红染色、碱性磷酸酶染色及RT-qPCR鉴定2D-Exo、3D-Exo对成骨分化能力的影响。结果与2D组比较,3D组细胞大小均等,聚拢成球形生长;钙黄绿素-AM/PI活死细胞双染可见3D培养的hUC-MSCs具有较高活性;转录组测序结果显示,与2D组比较,3D组上调基因富集于骨矿化、软骨发育、细胞外基质的组成、成骨细胞分化、血管生成、细胞增殖的正向调控及基因表达的正向调控等通路,下调基因富集于细胞增殖的负向调控、细胞迁移的负向调控、细胞凋亡等通路。透射电子显微镜观察可见两组外泌体的直径均为100 nm左右,呈现外泌体典型的杯状形貌特征,且均表达外泌体相关标志蛋白CD9、CD63和CD81。乳鼠颅骨成骨细胞染色结果显示,与2D-Exo组比较,3D-Exo组茜素红着色的钙结节数量及碱性磷酸酶染色强度均明显升高;RT-qPCR检测结果显示,与2D-Exo组比较,3D-Exo组成骨分化相关基因Bglap、Runx2、Alp、Col1a1、Spp1 mRNA相对表达量明显升高,差异有统计学意义(P<0.05)。结论3D培养的hUC-MSCs来源的外泌体可上调成骨相关基因Bglap、Runx2、Alp、Col1a1、Spp1的表达,相较于2D培养来源的外泌体具有更强的促成骨细�Objective To compare the differences between exosomes derived from three-dimensional(3D)cultured human umbilical cord-derived mesenchymal stem cells(hUC-MSCs)and conventional(2D)cultured hUC-MSCs in promoting osteoblast differentiation,and to explore the application possibility of exosomes derived from 3D-cultured hUC-MSCs in treatment related to promoting bone formation.Methods hUC-MSCs were divided into 2D group and 3D group,and cultured respectively.The morphological characteristics of the both groups were observed under optical microscope,and the viability of 3D-cultured hUC-MSCs was detected by calcein AM/PI double staining of living and dead cells.Transcriptome sequencing was performed to screen the differentially expressed genes between 2D group and 3D group,and GO enrichment analysis was conducted.Exosomes of 2D group and 3D group were extracted and divided into 2D-Exo group and 3D-Exo group.Characterization of exosomes was conducted by transmission electron microscope,nanoparticle tracking analysis and Western blotting.The exosomes excreted by 2D group and 3D group were applied to C57BL/6J mouse calvarial osteoblasts,respectively,and osteogenic differentiation was induced in vitro.The effects of exosomes cultured in 2D and 3D on osteogenic differentiation were identified by alizarin red staining,alkaline phosphatase staining and RT-qPCR.Results Comparing to 2D group,cells in 3D group were equal in size and grew into spherical shape.3D-cultured hUC-MSCs showed higher rate of viability verified by calcein AM/PI double staining of living and dead cells.Transcriptome sequencing results showed that the up-regulated genes in 3D group were enriched in bone mineralization,cartilage development,composition of extracellular matrix,osteoblast differentiation,angiogenesis,cell proliferation and gene expression.The down-regulated genes were enriched in negative regulation of cell proliferation,cell migration,and apoptotic process,etc.Transmission electron microscope observation showed that the exosome diameter in bot

关 键 词：三维立体培养 人脐带间充质干细胞 外泌体 成骨分化 

分 类 号：R318[医药卫生—生物医学工程]