CAJ | 학술논문

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。
목적：건립이가토위대상적태령기BMSC연구동물모형。방법통과인공수정방법，획득잉기3주적잉토4지，행부복산술취출태토20지，충취태토골수，이첩벽배양법진행체외확증배양。측량제3대태토BMSC적생장곡선，극륭형성솔，병진행성골、성지급성연골유도분화。령외，장원대세포동존30 d후복소，측량기제3대생장곡선，대동존전후증식능력적변화진행관찰。소묘전경관찰태토BMSC여β-TCP형성세포재료복합물적체외형태。장BMSCs-β-TCP복합물식입라서피하，우술후1、3、6개월분별취재，행HE、VG、Masson염색관찰。결과태토래원적BMSC우도치상차현미경하관찰，세포포만균균，정사형혹도삼각형상；전대후각대세포형태미발생명현변화，생장곡선상차불대；성골、성지이급성연골유도관찰도개결절、지방공포、점다당。동존30 d후복소，기제3대생장곡선여동존전상비미견명현변화。전경하관찰，여β-TCP복합7 d후，세포능균균긴밀첩합우재료상，신전량호분포균균。식입라서피불동시간점취재행HE、VG、Masson염색，균현시유신생골조직생성。결론이가토위연구동물모형，가이재태령기획취병분리득도BMSC，병가재이위구건조직공정골，시간충질간세포동물연구적신선택。
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.