大鼠骨内膜骨髓细胞增殖和成软骨分化能力的体外研究  

Study of Endosteal Bone Marrow Cells on Proliferation and Chondrogenic Differentiation in Rats in Vitro

作　　者：曾文波 兰生辉 陈美玲 袁功武 孙再杰 聂宇 刘曦明 ZENG Wenbo;LAN Shenghui;CHEN Meiling;YUAN Gongwu;SUN Zaijie;NIE Yu;LIU Ximing(Graduate School, Southern Medical University, Guangzhou Cruangdong 510515, China)

机构地区：[1]南方医科大学研究生院,广东广州510515 [2]解放军武汉总医院骨科湖北省骨创伤救治临床医学研究中心 [3]南方医科大学生物医学工程学院,广东广州510515 [4]解放军武汉总医院骨科 [5]陆军工程大学军械士官学校门诊部

出　　处：《华南国防医学杂志》2018年第4期215-220,共6页Military Medical Journal of South China

基　　金：国家自然科学基金资助项目(81601902);湖北省自然科学基金(2015CFB240;2014CFC1052);湖北省卫生计生委联合基金项目(WJ2018H0064);湖北省卫生计生西医类一般项目(WJ2015MB119);博士后科学基金(2015M572817)

摘　　要：目的对比骨内膜骨髓细胞(endosteal bone marrow cells,EBMCs)与中央骨髓细胞(center bone marrow cells,CBMCs)增殖及成软骨分化,为组织工程技术寻找较CBMCs更优良的种子细胞。方法取健康雄性SD大鼠的双侧股骨及胫骨,采用酶消化法分离EBMCs,采取密度梯度离心法分离CBMCs予体外增殖克隆,通过流式细胞术(flow cytometry,FCM)检测细胞表面分子CD29、CD34、CD44、CD45的表达。集落形成单位(colony forming unit,CFU)检测细胞克隆形成,5-溴脱氧尿嘧啶核苷(bromodeoxyuridine,Brd U)检测细胞增殖能力,定量反转录-聚合酶链反应(quantitative reverse transcription-polymerase chain reaction,qRT-PCR)检测细胞周期抑制因子相关基因p15、p16、p21、p27的表达。成软骨诱导培养21d后行阿尔新蓝染色并提取mRNA行qRT-PCR检测对比成软骨分化相关基因SOX9、aggrecan、Col2α1的表达。结果 EBMCs与CBMCs表面分子CD29、CD44均呈阳性表达,CD34、CD45均呈阴性表达,但EBMCs表达较CBMCs表达低,二者比较差异有统计学意义(P<0.05)。CFU检测中碱性磷酸酶(alkaline phosphatase,ALP)染色及结晶紫染色可见EBMCs比CBMCs颗粒沉淀数量更多,颗粒直径更大。Brd U检测细胞增殖能力,可见CBMCs的荧光信号倍增量(1.47±0.16),明显低于EBMCs的荧光信号倍增量(2.56±0.36),二者比较差异具有统计学意义(P<0.001)。细胞周期抑制因子p15、p16、p21、p27的表达,EBMCs中分别为0.315±0.052、0.158±0.023、0.416±0.051、0.100±0.022,明显低于CBMCs的1.000±0.000、0.481±0.064、1.342±0.135、0.461±0.156,二者比较差异有统计学意义(P<0.001)。阿尔新蓝染色可见EBMCs成软骨分化后染色蓝染较CBMCs更明显;EBMCs中成软骨骨分化相关基因SOX9,aggrecan,Col2α1的表达分别为1.783±0.305、0.918±0.201、0.384±0.108明显高于CBMCs的1.000±0.000、0.297±0.082、0.124±0.007,二者比较差异均具有统计学意义(P<0.001)。结论 EBMCs较CBMCs有更强的增殖分化能力,更适�Objective To compare the difference of proliferation and chondrogenic differentiation between endosteal bone marrow cells（EBMCs）and center bone marrow cells（CBMCs）,and find better seed cells for tissue engineering than CBMCs.Methods EBMCs and CBMCs were isolated from bilateral femur and tibia of healthy male SD rats by enzyme digestionand density gradient centrifugation,respectively. And CBMCs were given proliferation cloning in vitro.The expressions of cell surface molecules CD29,CD34,CD44 and CD45 were detected by flow cytometry（FCM）.Colony forming unit（CFU）was taken to detect the cell clone formation,5-Bromodeoxyuridine（BrdU）was used to detect the ability of cell proliferation,and quantitative reverse transcription-polymerase chain reaction（qRTPCR）was used to detect the expressions of gene p15,p16,p21,p27 associated with cell cycle inhibitors.After cultured with chondrocytes for 21 days,alcain blue wasused to stain,mRNA was extracted and compared among the expressions of chondrogenic differentiation related gene SOX9,aggrecan and Col2α1.Results The surface molecules CD29 and CD44 of EBMCs and CBMCs were both positive expressions,CD34 and CD45 were negative expressions,the expressions of EBMCs were lower than these of CBMCs（P0.05）.Alkaline phosphatase（ALP）staining and crystal violet staining in the CFU detection showed EBMCs had more precipitates and larger particles than CBMCs.BrdU was used to detect the cell proliferation ability.The proliferation of fluorescence signal in CBMCs,was significantly lower than that in EBMCs（1.47±0.16,vs.2.56±0.36,P0.001）.The expressions of cell cycle inhibitor p15,p16,p21 and p27 in EBMCs were significantly lower than those in CBMCs[（0.315±0.052,0.158±0.023,0.416±0.051,0.100±0.022）,vs.（1.000±0.000,0.481±0.064,1.342±0.135,0.461±0.156）,P0.001].Alcian blue staining showed the blue-staining in EBMCs after chondrogenic differentiation was much more obvious than that in CBMCs.The expressions of chondrogenic differentiation related

关 键 词：表皮生长因子受体 骨内膜骨髓细胞 中央骨髓细胞 成软骨分化 

分 类 号：Q332[生物学—遗传学]