CAJ | 학술논문

背景：骨髓间充质干细胞向软骨细胞定向诱导时间报道不一，对诱导中细胞动态变化进行观察比较的研究较少。 　　目的：观察兔骨髓间充质干细胞诱导8，11，14，17，20 d向成软骨细胞定向分化的动态变化情况以及诱导成熟时间。 　　方法：穿刺新西兰大白兔股骨穿刺抽取骨髓，密度梯度离心法分离、培养骨髓间充质干细胞。传至第3代后予以含转化生长因子β1等成分的无血清诱导液定向向软骨细胞诱导。根据不同的诱导时间分成为5组：8d组、11 d组、14 d组、17 d组及20 d组。通过观察各组细胞形态、甲苯胺蓝染色结果、Ⅱ型胶原免疫组化结果、培养液内多聚蛋白聚糖含量，比较各实验组定向诱导为成软骨细胞的情况。 　　结果与结论：诱导8 d时细胞形态有较明显改变，14 d时具有明显的软骨细胞形态。诱导培养液在诱导4 d时即可检出少量多聚蛋白聚糖，在8 d时浓度出现明显升高，并保持缓慢升高至20 d。在14 d时，诱导的细胞爬片甲苯胺蓝染色可见异染颗粒；Ⅱ型胶原免疫组化明显阳性表现。提示单层高密度接种的骨髓间充质干细胞，在转化生长因子β1等因子作用下能定向诱导为软骨细胞，诱导早期即可能有少量细胞向成软骨细胞分化，8 d时初步具有软骨细胞的形态、功能特征，14 d时成为较成熟的软骨细胞；诱导过程中细胞保持较高的生物活性。
배경：골수간충질간세포향연골세포정향유도시간보도불일，대유도중세포동태변화진행관찰비교적연구교소。 　　목적：관찰토골수간충질간세포유도8，11，14，17，20 d향성연골세포정향분화적동태변화정황이급유도성숙시간。 　　방법：천자신서란대백토고골천자추취골수，밀도제도리심법분리、배양골수간충질간세포。전지제3대후여이함전화생장인자β1등성분적무혈청유도액정향향연골세포유도。근거불동적유도시간분성위5조：8d조、11 d조、14 d조、17 d조급20 d조。통과관찰각조세포형태、갑분알람염색결과、Ⅱ형효원면역조화결과、배양액내다취단백취당함량，비교각실험조정향유도위성연골세포적정황。 　　결과여결론：유도8 d시세포형태유교명현개변，14 d시구유명현적연골세포형태。유도배양액재유도4 d시즉가검출소량다취단백취당，재8 d시농도출현명현승고，병보지완만승고지20 d。재14 d시，유도적세포파편갑분알람염색가견이염과립；Ⅱ형효원면역조화명현양성표현。제시단층고밀도접충적골수간충질간세포，재전화생장인자β1등인자작용하능정향유도위연골세포，유도조기즉가능유소량세포향성연골세포분화，8 d시초보구유연골세포적형태、공능특정，14 d시성위교성숙적연골세포；유도과정중세포보지교고적생물활성。
BACKGROUND:The reported time of bone marrow mesenchymal stem cel s induced to differentiate into chondrocytes is different. Few studies have observed and compared the cel s’ dynamic transformation during the induction process. OBJECTIVE:To observe the dynamic differentiation and the mature time of rabbit bone marrow mesenchymal stem cel s which were directional y induced to chondroblasts for 8, 11, 14, 17, 20 days. METHODS:Bone marrow was aspirated from the femur of New Zeal rabbits, and bone marrow mesenchymal stem cel s were isolated by gradient centrifugation. After cultivation and amplification, bone marrow mesenchymal stem cel s at passage 3 were directional y induced to chondrocytes by the serum-free medium containing transforming growth factor beta-1. The experiments were divided into five groups according to different induction time points:8 days, 11 days, 14 days, 17 days, 20 days. Then cel ular morphology, toluidine blue staining, typeⅡ col agen immunohistochemistry, aggrecan content in induction medium, and chondrogenic differentiation in each group were observed and compared. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cel s had apparently transformed in morphology at 8 days of induction, and presented obvious chondrocytes’ morphology at 14 days. The aggrecan in induction medium could be detected at a low level at 4 days, significantly increased at 8 days, and maintained slow increasing at 20 days. At 14 days, the metachromatic particles could be found by toluidine blue staining, and the col agen type Ⅱimmunohistochemistry was significantly positive in cel climbing slice. Experimental findings indicate that, bone marrow mesenchymal stem cel s that are monolayer cultured in a high density can be induced into chondroblasts at the effect of transforming growth factor beta-1 and other factors. There are a few chondroblasts in the early induction process, then cel s begin to have chondrocytes morphology and function after induced for 8 days, and may differentiate to mature chondrocytes at 14 days. In addition, they can keep a high biological activity in the induction process.