应变诱导BMSCs腱向分化的实验研究  

STRAIN-INDUCED TENOGENIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS

作　　者：廖梅旭 宁良菊[1] 陈晓禾[1] 李秀群[1] 罗静聪[1] 秦廷武[1] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室.干细胞与组织工程研究室,成都610041 [2]四川省人民医院辅助生殖中心

出　　处：《中国修复重建外科杂志》2010年第7期817-821,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30570469);教育部留学回国人员科研启动基金资助项目[(2010)609]~~

摘　　要：目的研究大鼠BMSCs与小肠黏膜下层(small intestinal submucosa,SIS)复合后,施加动态应变刺激能否使BMSCs在体外分化为肌腱细胞(tenocytes,TCs)。方法取1周龄SD大鼠骨髓,采用贴壁培养法分离培养大鼠BMSCs,并用多向分化诱导法和流式细胞仪检测进行鉴定。用生物力学试验机行SIS的拉伸破坏实验,计算SIS的弹性应变。将分离纯化后的第2代BMSCs以2.5×105个/cm2细胞密度种植于3cm×1cm大小的SIS上,形成BMSCs-SIS复合物,固定于应变培养装置对其进行静态培养2d后,施加应变刺激(拉伸频率为0.02Hz,作用时间为15min/h、12h/d,应变幅度为5%)动态培养5d,作为实验组;BMSCs-SIS复合物持续静态培养作为对照组;采用组织块法分离培养SD大鼠鼠尾TCs,用第2代TCs与SIS复合,相同条件下静态培养作为阳性对照组。扫描电镜观察应变培养后BMSCs形态变化,ELISA法检测培养后细胞上清液中2种TCs标志蛋白Scleraxis和Tenomodulin的含量。结果分离培养的SD大鼠BMSCs经多向分化诱导后,可以分化为成骨细胞和脂肪细胞,且流式细胞仪检测示表面标志抗原CD34、CD45为阴性,CD90为阳性,符合BMSCs生物学特性。SIS拉伸破坏实验结果显示,SIS平均弹性应变为39.5%。扫描电镜观察示实验组材料上细胞具有类TCs形态;ELISA法检测示,实验组应变培养后细胞上清液中Scleraxis和Tenomodulin含量分别为(3.56±0.91)μmol/L和(4.27±1.10)μmol/L,阳性对照组分别为(14.73±2.30)μmol/L和(10.65±1.51)μmol/L,对照组分别为(0.23±0.14)μmol/L和(0.16±0.10)μmol/L;3组比较差异均有统计学意义(P<0.05)。结论适当的应变刺激培养可在体外诱导BMSCs向TCs方向分化,尚需进一步研究最佳的应变刺激诱导条件。Objective To study the possibility of bone marrow mesenchymal stem cells（BMSCs） differentiation into tenocytes（TCs） under strain stimulation by co-culture of BMSCs-small intestinal submucosa（SIS） composites in vitro.Methods BMSCs were isolated by adherent culture from the bone marrow of 1-week-old SD rats.Inducing method of multiple differentiation and flow cytometry were applied to identify the cells.The stress-strain curve of SIS was measured with Instron machine.Purified BMSCs（2nd passage,2.5 × 105 cells/cm2） were seeded on SIS（3 cm × 1 cm at size） and cultured for 2 days and then continued for another 5 days under strain stimulation（stretching frequency was 0.02 Hz,action time was 15 minutes/hour and 12 hours/day,strain amplitude was 5%） as experimental group,while the BMSCs-SIS composites were sustained static culture as control group.TCs were isolated from tail of 1-week-old SD rats.TCs-SIS composites were cultured under non-strained as positive control group.Scanning electron microscope（SEM） was used to examine the morphological changes of BMSCs after strain stimulation.The contents of Scleraxis and Tenomodulin in supernatant were tested by ELISA kit.Results The BMSCs could be induced to differentiate into osteoblasts and lipocytes,and showed the results of CD34-,CD45-,and CD90＋,which were accorded with the biological characteristics of BMSCs.The failure test of SIS showed that the average elastic strain was 39.5%.SEM observation showed that the strain-stimulated BMSCs had the TCs-like morphological characteristics.The contents of Scleraxis and Tenomodulin in supernatant of experimental group,control group,and positive control group were（3.56 ± 0.91） μmol/L and（4.27 ± 1.10） μmol/L,（0.23 ± 0.14） μmol/L and（0.16 ± 0.10） μmol/L,and（14.73 ± 2.30） μmol/L and（10.65 ± 1.51） μmol/L,respectively.There were significant differences among 3 groups（P 0.05）.Conclusion Appropriate strain stimulation could induce BMSCs differentiate into TCs,and the b

关 键 词：组织工程 BMSCS 肌腱细胞 应变刺激 

分 类 号：R318[医药卫生—生物医学工程] Q66[医药卫生—基础医学]