CAJ | 학술논문

背景：体外分离培养出生长状态好、高纯度、增殖能力强和数量充足的大鼠骨髓间充质干细胞，是将其作为种子细胞用于组织和细胞移植的重要前提。 　　目的：建立简便、快速、有效的SD大鼠骨髓间充质干细胞体外分离培养方法，并观察其生物学特性。 　　方法：采用全骨髓法将 SD 大鼠双侧股骨和胫骨骨髓细胞进行体外分离培养，贴壁接种法进行细胞纯化、传代。观察细胞生长形态及特征，绘制细胞生长曲线，检测细胞表面标记物，采用体外诱导剂诱导细胞分别向成骨、成软骨、成脂方向分化。 　　结果与结论：全骨髓贴壁接种法分离培养的骨髓间充质干细胞生长旺盛、纯度高，细胞生长形态呈长梭形，极性排列，细胞生长呈S形生长曲线，群体倍增时间为29 h，细胞在连续传10代后仍具有较强的增殖能力。第3代骨髓间充质干细胞的表面标记物CD44、CD29、CD90均呈阳性表达，CD45、CD34、CD11b则呈阴性表达。第3代骨髓间充质干细胞分别经成骨、成软骨、成脂诱导剂诱导后，茜素红染色、碱性磷酸酶染色、von-kossa矿化结节染色、甲苯胺蓝染色和油红O染色均呈阳性。结果验证全骨髓贴壁接种法是一种简便可靠的体外分离培养方法，能获得纯度较高的骨髓间充质干细胞，经实验鉴定第3代骨髓间充质干细胞生物活性最佳，且具有多向诱导分化能力，适合作为后续实验的种子细胞。
배경：체외분리배양출생장상태호、고순도、증식능력강화수량충족적대서골수간충질간세포，시장기작위충자세포용우조직화세포이식적중요전제。 　　목적：건립간편、쾌속、유효적SD대서골수간충질간세포체외분리배양방법，병관찰기생물학특성。 　　방법：채용전골수법장 SD 대서쌍측고골화경골골수세포진행체외분리배양，첩벽접충법진행세포순화、전대。관찰세포생장형태급특정，회제세포생장곡선，검측세포표면표기물，채용체외유도제유도세포분별향성골、성연골、성지방향분화。 　　결과여결론：전골수첩벽접충법분리배양적골수간충질간세포생장왕성、순도고，세포생장형태정장사형，겁성배렬，세포생장정S형생장곡선，군체배증시간위29 h，세포재련속전10대후잉구유교강적증식능력。제3대골수간충질간세포적표면표기물CD44、CD29、CD90균정양성표체，CD45、CD34、CD11b칙정음성표체。제3대골수간충질간세포분별경성골、성연골、성지유도제유도후，천소홍염색、감성린산매염색、von-kossa광화결절염색、갑분알람염색화유홍O염색균정양성。결과험증전골수첩벽접충법시일충간편가고적체외분리배양방법，능획득순도교고적골수간충질간세포，경실험감정제3대골수간충질간세포생물활성최가，차구유다향유도분화능력，괄합작위후속실험적충자세포。
BACKGROUND:Tissue and cellimplantation entails high-quality seed cells. In order to satisfy this requirement, it is crucial to produce adequate wel-conditioned, high-purity and strong proliferation ability bone marrow-derived mesenchymal stem cells. OBJECTIVE:To establish a simple, rapid and effective in vitro isolation and culture method of bone marrow-derived mesenchymal stem cells, and to define the biological features of bone marrow mesenchymal stem cells. METHODS:Rat bone marrow mesenchymal stem cells were isolated from the bilateral tibial and femoral bones by the method of whole bone marrow, then purified and passaged by attachment method. The morphology and features of bone marrow mesenchymal stem cells were observed, the growth curve was drawn and the cellsurface antigen was detected by flow cytometry. The bone marrow mesenchymal stem cells were induced to differentiate along the osteogenic, chondrogenic and adipogenic lineages. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells isolated by the whole bone marrow adherence method grew vigorously and were highly purified. The cultured cells were spindle-shaped. The growth curve was S-shaped and the population doubling time was 29 hours. The cells stil maintained a strong proliferative capacity after they were passaged for 10 generations. The surface markers such as CD44, CD29, CD90 were positive, while CD45, CD34, CD11b were negative. At the third passage, bone marrow mesenchymal stem cells were induced to differentiate along the osteogenic, chondrogenic and adipogenic lineages, respectively. Fol owing induction, Alizarin red staining, alkaline phosphatase staining, von-kossa mineralized nodules staining, toluidine blue staining, and oil red O staining were al positive. This shows that the whole bone marrow adherence method is a simple and reliable method for the in vitro isolation, culture and proliferation of bone marrow mesenchymal stem cells. Moreover, they have multi-lineage differentiation capacity under different inducers. The third passage bone marrow mesenchymal stem cells have the highest biological activity and can act as the ideal seed cells for subsequent experiments.