Gefitinib对骨外膜间充质干细胞和骨髓间充质干细胞生物学功能的影响  

Effects of Gefitinib on the Biological Functions of Periosteum Mesenchymal Stem Cells and Bone Marrow Mesenchymal Stem Cells

作　　者：王华松 兰生辉 杨晨曦 丰瑞兵 庞炯宇 袁功武 曾文波 WANG Huasong;LAN Shenghui;YANG Chenxi;FENG Ruibing;PANG Jiongyu;YUAN Gongwu;ZENG Wenbo(Department of Orthopaedics,General Hospital of Central Theatre Command,Wuhan Hubei 430070,China)

机构地区：[1]中部战区总医院骨科,湖北武汉430070 [2]湖北省中西医结合医院骨科 [3]重庆医科大学附属第三医院骨科

出　　处：《华南国防医学杂志》2020年第5期295-300,322,共7页Military Medical Journal of South China

基　　金：国家自然科学基金(81601902);湖北省卫生健康委员会联合基金(WJ2019H097)。

摘　　要：目的探讨表皮生长因子受体(epidermal growth factor receptor,EGFR)信号通路抑制剂Gefitinib对骨外膜间充质干细胞(periosteum mesenchymal stem cells,PMSCs)和骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)生物学功能的影响。方法将10只健康雄性SD大鼠随机分为实验组(n=5)和对照组(n=5),均予以制备股骨闭合骨折模型。实验组给予Gefitinib灌胃(100mg/kg·d),对照组给予相同剂量甲基纤维素灌胃。术后1周在麻醉状态下处死大鼠并分离提取股骨BMSCs和PMSCs,通过流式细胞术鉴定细胞表面分化抗原群(cluster of differentiation,CD)29、CD34、CD45、CD90;成纤维样细胞集落形成单位(colony forming unit-fibroblast,CFU-F)检测形成沉淀的碱性磷酸酶(alkaline phosphatase,ALP)染色阳性细胞数及沉淀直径大小;5-溴-2-脱氧尿苷(5-Bromo-2-deoxyuridine,BrdU)标记法检测细胞增殖能力;实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,qPCR)检测细胞周期抑制因子基因(p15、p16、p21、p27)mRNA的表达。成骨诱导后行Von Kossa染色观察、成软骨诱导分化后行阿尔新蓝染色观察,qPCR法检测成骨分化基因(osteocalcin、bsp、osterix、RUNX2)mRNA和成软骨分化基因(Aggrecan、Col2a1、Sox9)的表达情况。结果PMSCs和BMSCs的CD29和CD90均呈阳性表达,CD34和CD45均呈阴性表达。染色观察可见PMSCs形成沉淀的ALP阳性细胞数多于BMSCs,PMSCs沉淀直径大于BMSCs。Gifitinib阻断EGFR信号后,BrdU检测显示实验组的BrdU值低于对照组(P<0.05),且PMSCs的BrdU值高于BMSCs(P<0.05);实验组细胞周期抑制因子相关基因表达水平低于对照组,且PMSCs相关基因的表达水平明显低于BMSCs(P<0.05);实验组成骨分化及成软骨分化相关基因的表达水平高于对照组(P<0.05),且PMSCs相关基因的表达水平明显高于BMSCs(P<0.05)。结论EGFR信号通路抑制剂Gifitinib抑制PMSCs和BMSCs的增殖,但促进其成骨及成软骨分化。Objective To investigate the effects of signal pathway inhibitor Gefitinib of epidermal growth factor receptor(EGFR)on the biological functions of periosteum mesenchymal stem cells(PMSCs)and bone marrow mesenchymal stem cells(BMSCs).Methods Ten healthy male SD rats were randomly divided into experimental group(n=5)and control group(n=5),and all of them were made into closed fracture model of femur.The rats in experimental group were given Gefitinib(100mg/kg·d)by gavage,and the control group was given the same dose of methylcellulose by gavage.The BMSCs and PMSCs of femur were isolated and extracted in anesthetized rats one week after operation.The cell surface cluster of differentiation 29(CD29),CD34,CD45and CD90were identified by flow cytometry.The deposition of alkaline phosphatase(ALP)stained positive cell number and sedimentation diameter were detected by colony forming unitfibroblast(CFU-F);the cell reproductive capacity was detected by 5-Bromo-2-deoxyuridine(BrdU)assay.The expression of cell cycle inhibitor genes(p15,p16,p21,p27)mRNA were detected by quantitative real-time polymerase chain reaction(qPCR).After osteogenesis induction was observed by Von Kossa staining,and after chon-drogenic differentiation induction was observed by Alxin blue staining.The expression of osteogenesis differentiation genes(costeocalcin,bsp,osterix,RUNX2)mRNA and chondrogenic differentiottion genes(Aggrecan,Col2al,Sox9)were detected by qPCR.Results The expression of CD29and CD90in PMSCs and BMSCs were positive,while the expression of CD34and CD45were negative.The number of ALP positive cell precipitated of PMSCs was more than that of BMSCs,and the diameter of PMSCs precipitated was larger than that of BMSCs.The EGFR signal was blocked by Gifitinib,BrdU detection showed that the BrdU value of experimental group was lower than that of control group(P<0.05),and BrdU value of PMSCs was higher than that of BMSCs(P<0.05);the expression of cell cycle inhibitor related genes in experimental group were lower than those in control group

关 键 词：吉非替尼 表皮生长因子受体 间充质干细胞 成骨分化 成软骨分化 

分 类 号：R332[医药卫生—人体生理学] R681.3[医药卫生—基础医学]