EphB/EphrinB信号通路对成骨分化能力的影响  

Effect of EphB/EphrinB signaling pathway on the osteogenic differentiation

作　　者：刘军[1] 李旭升[1] 甄平 田琦 周胜虎[1] 王伟[1] 李玉娟 何晓乐 LIU Jun;HE Xiaole;LI Yujuan;ZHEN Ping;TIAN Qi;ZHOU Shenghu;WANG Wei;LI Xusheng(Department of Joint Surgery,Institute of Orthopedics,the 940th Hospital of PLA Joint Logistics Support Force,Lanzhou 730000,China;Department of Surgical anesthesiology,the 940th Hospital of PLA Joint Logistics Support Force,Lanzhou 730000,China;Department of Gerontology,Xijing Hospital,the Air Force Military Medical University,Xi’an 710032,China)

机构地区：[1]中国人民解放军联勤保障部队940医院(原兰州军区兰州总医院)全军骨科中心,甘肃兰州730050 [2]中国人民解放军联勤保障部队940医院(原兰州军区兰州总医院)手麻科,甘肃兰州730050 [3]空军军医大学西京医院老年病科,陕西西安710032

出　　处：《中国骨质疏松杂志》2020年第3期345-352,共8页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金(81371983);全军后勤科研计划面上项目(CWH17J009);全军医学科技青年培育计划(19QNP047);甘肃省青年科技基金(1606RJYA300);甘肃省卫生行业科研计划项目(GSWSKY2018-21);甘肃省自然科学基金(1606RJZA208)。

摘　　要：目的探讨EphB/EphrinB信号通路对去势小鼠骨髓间充质干细胞(bone marrow derived mesenchymal stem cells,BMSCs)的成骨分化能力和绝经后骨质疏松动物模型骨量的影响。方法将24只8周龄健康雌性BALB/c小鼠随机分为去势组(OVX组)和假手术组(Sham组),检测小鼠BMSCs中EphB/EphrinB信号通路表达水平。(2)将去势小鼠分为4组,分别进行腹腔注射相应Eph受体激动剂:EfnB1-Fc及EfnB2-Fc,Eph受体抑制剂EphB2-Fc,对照组腹腔注射human IgG-Fc。各组分别于术后10周将小鼠脱颈处死,在Micro-CT分析下比较股骨骨质情况。去势组和假手术组小鼠取股骨与胫骨骨髓,经密度梯度离心法分离培养并鉴定其骨髓间充质干细胞至P3用于实验,各组BMSCs在成骨诱导条件下7 d后,通过Realtime PCR检测成骨相关基因(Runx2、ALP、Osterix)及破骨细胞分化相关基因(OPG、RANKL)的表达水平,成骨分化14、21 d后,采用ALP染色和茜素红染色观察成骨分化能力。结果与假手术组(Sham组)相比较,去势组(OVX组)中EfnA2、EphB4、EfnB2、EfnB1及EphA4表达显著增高(P<0.01);Sham组中EphA2及EfnB2表达显著降低(P<0.01)。10周后Micro-CT结果显示,与去势对照Fc组比较,Sham组、EfnB1-Fc、EfnB2-Fc及EphB2-Fc去势组骨小梁结构均较完整、骨小梁密度较好(P<0.01);与EfnB1-Fc、EfnB2-Fc去势组相比较,Sham组和EphB2-Fc去势组的骨密度及骨体积分数均明显增高(P<0.01)。Realtime PCR检测成骨相关基因提示,与去势对照Fc组比较,EfnB1-Fc、EfnB2-Fc和EphB2-Fc去势组中ALP与Osterix的表达明显升高(P<0.01);EfnB1-Fc和EphB2-Fc可显著提高BMSCs成骨分化中ALP的活性和骨基质矿化能力,以EphB2-Fc效果最为显著(P<0.01)。与去势对照Fc组比较,EfnB1-Fc、EfnB2-Fc和EphB2-Fc去势组中RANKL的表达未见明显差异(P>0.05),但OPG的表达明显升高(P<0.01),其中,EfnB1-Fc去势组与EphB2-Fc去势组中OPG/RANKL的比率升高最为明显(P<0.01)。结论EphB2/EfnB1双向信号通路可逆转Objective To explore the effect of EphB/EphrinB signaling pathway on the osteogenic differentiation of mouse bone marrow derived mesenchymal stem cells(BMSCs)and the postmenopausal osteoporosis animal model in mice.Methods 1)Twenty-four healthy 8-week-old BALB/c mice were randomly divided into castration group(OVX group)and sham operation group(Sham group).The expression level of Eph/Ephrin signaling pathway in BMSCs was detected.2)The ovariectomized mice were divided into 4 groups.The Eph receptor agonists EfnB1-Fc and EfnB2-Fc,Eph receptor inhibitor EphB2-Fc,and human IgG-Fc as control,were intraperitoneally injected,respectively.The mice were sacrificed by cervical dislocation 10 weeks after the surgery.The femoral bones were compared under micro-CT analysis.The bone marrow of the femurs and the tibia was extracted from the ovariectomized and sham-operated mice.BMSCs were isolated and cultured by density gradient centrifugation and identified to P3.After osteogenic induction for 7 days,the expression levels of Runx2,ALP,Osterix,OPG,and RANKL were detected using realtime PCR.After 14 days and 21 days of osteogenic differentiation,the ability of osteogenic differentiation was observed with ALP staining and Alizarin red staining.Results Compared with those in the OVX group,the expressions of EfnA2,EphB4,EfnB2,EfnB1,and EphA4 in the Sham group increased significantly(P<0.01),and the expression difference was similar to those in humans.The expressions of EfnA2 and EfnB2 reduced significantly in the Sham group(P<0.01).After 10 weeks,the result of micro-CT showed that compared with the control group,the trabecular structure in the Sham group,and EfnB1-Fc,EfnB2-Fc,and EphB2-Fc in the castration group was relatively intact and the trabecular bone density was better(P<0.01).Compared with those in EfnB1-Fc and EfnB2-Fc castration groups,the bone mineral density and bone volume fraction in the Sham group and EphB2-Fc castration group increased significantly(P<0.01).The result of real-time PCR for detection of osteogenesis

关 键 词：EphB/EphrinB信号通路 去势小鼠 骨髓间充质干细胞 成骨分化 

分 类 号：R684.3[医药卫生—骨科学]