依诺肝素对骨髓间充质干细胞成骨过程及其分泌外泌体的影响  

Effects of enoxaparin on osteogenic differentiation of bone marrow stem cells and their exosomes

作　　者：范伟[1] 李岩[1] 李春江[1] 栗树伟[1] 刘亮[1] 王斌[1] 王东[1] 孙海钰[1] Fan Wei;Li Yan;Li Chunjiang;Li Shuwei;Liu Liang;Wang Bin;Wang Dong;Sun Haiyu(Second Hospital of Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学第二医院,太原030001

出　　处：《中华创伤骨科杂志》2020年第7期618-623,共6页Chinese Journal of Orthopaedic Trauma

基　　金：国家自然科学基金(81802204)。

摘　　要：目的:研究依诺肝素对骨髓间充质干细胞(BMSCs)成骨分化过程及其分泌外泌体的影响。方法:提取4周龄雄性SD大鼠的BMSCs,对其表面抗原和多系分化潜能进行鉴定,诱导其向成骨方向分化。10 IU/mL依诺肝素处理BMSCs 14 d后,采用试剂盒法提取BMSCs分泌的外泌体,运用透射电镜观察外泌体形态,检测外泌体CD63表达,茜素红染色分析BMSCs的成骨化,分别检测BMSCs及外泌体中成骨相关蛋白骨钙素及骨形态发生蛋白-2(BMP-2)的表达水平。结果:分离培养的BMSCs呈长梭形,形态均匀一致,高表达CD29和CD44,低表达CD34和CD45,符合间充质干细胞特征;提取外泌体成椭圆形,直径30~80 nm,表达CD63。茜素红染色提示依诺肝素处理组矿化结节数较空白对照组低。依诺肝素处理组BMSCs骨钙素[(48.81±8.23)ng/mL]和BMP-2[(311.45±27.59)pg/mL]含量低于空白对照组[(80.43±10.74)ng/mL和(399.23±32.25)pg/mL],差异均有统计学意义(P<0.05)。而相同BMSCs细胞数量来源的依诺肝素干预下的外泌体中骨钙素[(1.45±0.15)ng/mL]和BMP-2[(18.47±0.54)pg/mL]含量高于空白对照组[(1.00±0.12)ng/mL]和(9.07±0.36)pg/mL],差异均有统计学意义(P<0.05)。结论:依诺肝素处理可以抑制BMSCs成骨化过程,其机制可能与依诺肝素直接抑制BMSCs成骨相关蛋白骨钙素及BMP-2的表达以及间接通过诱导BMSCs中的骨钙素及BMP-2以外泌体形式排出从而进一步减少BMSCs成骨相关蛋白水平有关。Objective To study the effects of enoxaparin on osteogenic differentiation of bone marrow stem cells(BMSCs)and the exosomes derived from BMSCs.Methods After the BMSCs from 4-week old male SD rats were cultured,their surface antigen and multilineage differentiation potentials were identified.Subsequently,the BMSCs were incubated with osteogenic differentiation medium containing 10 IU/mL enoxaparin for 14 days.After the exosomes derived from BMSCs(BMSC-Exos)were extracted by the kit method,their structure was observed by transmission electron microscopy and their surface antigen CD63 detected by Western blot.Alizarin red staining was used to analyze the osteogenic differentiation of BMSCs.Osteogenic proteins including OCN and BMP-2 in BMSCs and exosomes were detected.Results The spindle-shaped BMSCs isolated and cultured showed a uniform spiral pattern.They expressed highly the surface markers CD29 and CD44 but lowly CD34 and CD45,indicating that the majority of the cells were BMSCs.BMSC-Exos,in an oval shape with a diameter of about 30 to 80 nm,expressed CD63.Alizarin red staining showed that the number of mineralized nodules in the enoxaparin treatment group was significantly lower than that in the control group.Western blot analysis indicated that enoxaparin inhibited the expression of osteocalcin(OCN)and BMP-2 in BMSCs.ELISA results showed that the protein levels of OCN(48.81 ng/mL±8.23 ng/mL)and BMP-2(311.45 pg/mL±27.59 pg/mL)in the BMSCs treated with enoxaparin were significantly lower than those of OCN(80.43 ng/mL±10.74 ng/mL)and BMP-2(399.23 pg/mL±32.25 pg/mL)in the control BMSCs(P<0.05).The contents of OCN(1.45 ng/mL±0.15 ng/mL)and BMP-2(18.47 pg/mL±0.54 pg/mL)in the exosomes from BMSCs treated with enoxaparin were significantly higher than those of OCN(1.00 ng/mL±0.12 ng/mL)and BMP-2(9.07 ng/mL±0.36 pg/mL)in the exosomes from control BMSCs(P<0.05).Conclusions Enoxaparin may inhibit the osteogenic differentiation of BMSCs.The mechanism of this might be related to its direct inhibition against the e

关 键 词：依诺肝素 骨髓细胞 骨钙素 成骨分化 外泌体 

分 类 号：R68[医药卫生—骨科学]