组织工程与重建外科杂志

组织工程与重建外科杂志 ›› 2015, Vol. 11 ›› Issue (2): 99-103.doi: 10.3969/j.issn.1673-0364.2015.02.013

• 论著 • 上一篇    下一篇

家兔作为胎龄期骨髓间充质干细胞动物模型的实验研究

周权，祁美武，张智勇，周广东，叶金艳，周晓   

  1. 南华大学；上海交通大学医学院附属第九人民医院整复外科, 上海市组织工程研究重点实验室；石埠子镇卫生院；湖南省肿瘤医院整形外科
  • 发布日期:2020-07-23

Rabbit as Animal Model of Fetal Bone Mesenchymal Stem Cells

ZHOU Quan,QI Meiwu,ZHANG Zhiyong,ZHOU Guangdong,YE Jinyan,ZHOU Xiao   

  1. University of South China; Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine;Shanghai Key Laboratory of Tissue Engineering;Shi Bu Zi Town Health Center; Department of Plastic Surgery, Hunan Cancer Hospital;
  • Published:2020-07-23

摘要： 目的建立以家兔为对象的胎龄期BMSC研究动物模型。方法通过人工授精方法,获得孕期3周的孕兔4只,行剖腹产术取出胎兔20只,冲取胎兔骨髓,以贴壁培养法进行体外扩增培养。测量第3代胎兔BMSC的生长曲线,克隆形成率,并进行成骨、成脂及成软骨诱导分化。另外,将原代细胞冻存30 d后复苏,测量其第3代生长曲线,对冻存前后增殖能力的变化进行观察。扫描电镜观察胎兔BMSC与β-TCP形成细胞材料复合物的体外形态。将BMSCs-β-TCP复合物植入裸鼠皮下,于术后1、3、6个月分别取材,行HE、VG、Masson染色观察。结果胎兔来源的BMSC于倒置相差显微镜下观察,细胞饱满均匀,呈梭形或倒三角形状;传代后各代细胞形态未发生明显变化,生长曲线相差不大;成骨、成脂以及成软骨诱导观察到钙结节、脂肪空泡、黏多糖。冻存30 d后复苏,其第3代生长曲线与冻存前相比未见明显变化。电镜下观察,与β-TCP复合7 d后,细胞能均匀紧密贴合于材料上,伸展良好分布均匀。植入裸鼠皮不同时间点取材行HE、VG、Masson染色,均显示有新生骨组织生成。结论以家兔为研究动物模型,可以在胎龄期获取并分离得到BMSC,并可在异位构建组织工程骨,是间充质干细胞动物研究的新选择。

关键词: 骨髓间充质干细胞, 种子细胞, 骨组织工程, 家兔

Abstract: Objective To construct an animal model of fetal bone mesenchymal stem cells with rabbit. Methods Four pregnant rabbits (pregnancy=3 weeks) were treated with artificial fertilization, and 20 fetal rabbits were obtained with cesarean section. Fetal rabbit bone marrow was harvested and cultured in vitro. The cell morphology, growth curve, cloning efficiency and trilinear differentiation of BMSCs of passage 3 were observed. Cryopreserved primary cells were recovered after 30 days, the growth curve of passage 3 was measured to compare with that non-cryopreserved. With scanning electron microscope, the fetal cells in β-TCP cell-material compound were observed. Finally, after osteogenic induction, the BMSCs-β-TCP compound was implanted into the nude mice subcutaneously and the histological results of tissue-engineered bone at 1, 3, 6 months were observed. Results The BMSCs from fetal rabbit demonstrated a spindle-shaped morphology. After several passages, the morphology and growth curve remained almost the same. The calcium, fat and mucopolysaccharide were witnessed in trilinear differentiation. After cryopreserved for 30 days, the growth curve of passage 3 showed few changes compared with non-cryopreserved. According to SEM, the fetal cells tightly attached to the materials and extended well after seeded on β-TCP for 7 days. The fresh bone tissue were all observed at different time point by histological staining. Conclusion Rabbit as animal model can isolate and procure the fetal BMSCs and has good osteogenic ability in vivo. And it can be the new choice of the animal model for fetal bone mesenchymal stem cells.

Key words: Bone marrow mesenchymal stem cells, Seed cells, Bone tissue engineering, Rabbit

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