CAJ | 학술논문

背景：骨髓间充质干细胞是理想的组织工程种子细胞来源，但是不同年龄的骨髓间充质干细胞体外增殖、分化特点有很大差异，有关年龄与骨髓间充质干细数量的关系目前尚缺少系统研究。目的：观察不同鼠龄大鼠骨髓间充质干细胞诱导分化活性的差异。方法：通过全骨髓贴壁培养法，体外分离、纯化、扩增 SD 大鼠骨髓间充质干细胞，倒置相差显微镜观察形态学特点，流式细胞仪检测细胞表面标记，体外诱导骨髓间充质干细胞向成骨细胞、成软骨细胞分化并鉴定。取第3代2，4，6，8，12周龄和10，12月龄大鼠骨髓间充质干细胞成骨诱导1，2，3周，采用酶联免疫法检测骨钙素含量。结果与结论：体外培养的骨髓间充质干细胞为贴壁生长，长梭形成纤维细胞样细胞，可增殖形成克隆；流式细胞仪分析骨髓间充质干细胞CD29、CD90表达阳性，CD45表达阴性，CD44部分表达；经成骨分化诱导，细胞碱性磷酸酶染色阳性，茜素红染色阳性；经成软骨分化诱导，阿利辛蓝染色阳性，可见通过全骨髓贴壁法可成功地从大鼠骨髓中分离出骨髓间充质干细胞。通过不同鼠龄骨髓间充质干细胞诱导成骨分化后骨钙素含量测定得出骨髓间充质干细胞增殖分化能力随鼠龄的增长而下降。
배경：골수간충질간세포시이상적조직공정충자세포래원，단시불동년령적골수간충질간세포체외증식、분화특점유흔대차이，유관년령여골수간충질간세수량적관계목전상결소계통연구。목적：관찰불동서령대서골수간충질간세포유도분화활성적차이。방법：통과전골수첩벽배양법，체외분리、순화、확증 SD 대서골수간충질간세포，도치상차현미경관찰형태학특점，류식세포의검측세포표면표기，체외유도골수간충질간세포향성골세포、성연골세포분화병감정。취제3대2，4，6，8，12주령화10，12월령대서골수간충질간세포성골유도1，2，3주，채용매련면역법검측골개소함량。결과여결론：체외배양적골수간충질간세포위첩벽생장，장사형성섬유세포양세포，가증식형성극륭；류식세포의분석골수간충질간세포CD29、CD90표체양성，CD45표체음성，CD44부분표체；경성골분화유도，세포감성린산매염색양성，천소홍염색양성；경성연골분화유도，아리신람염색양성，가견통과전골수첩벽법가성공지종대서골수중분리출골수간충질간세포。통과불동서령골수간충질간세포유도성골분화후골개소함량측정득출골수간충질간세포증식분화능력수서령적증장이하강。
BACKGROUND:Bone marrow mesenchymal stem cells are ideal as tissue engineering seed cells, but the proliferation and differentiation of mesenchymal stem cells in vitro is very different at different ages. Moreover, there are few reports on the association between age and the number of bone marrow mesenchymal stem cells. OBJECTIVE:To observe the difference in differentiation ability of bone marrow mesenchymal stem cells from rats at different ages. METHODS:We isolated, purified and amplified the mesenchymal stem cells from rat bone marrow in vitro by the whole bone marrow adherent culture;observed the morphological characteristics of mesenchymal stem cells under an inverted phase contrast microscope;detected cellsurface markers by flow cytometer. Then, mesenchymal stem cells were induced in vitro into osteoblasts and chondroblasts and verified. Passage 3 cells from rats at ages of 2, 4, 6, 8, 12 weeks and 10, 12 months were subjected to osteogenic induction at weeks 1, 2, 3. ELISA was used to determine osteocalcin content. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells cultured in vitro were adherent and exhibited a fibroblast-like spindle shape. In vitro, cells proliferated quickly to form colonies. Flow cytometry showed that the cells were positive for CD29, CD90, but negative for CD45, and partial y expressed CD44. After osteogenic induction, cells were positive for alkaline phosphatase staining and alizarin red staining;after chondrogenic induction, cells were positive for alcian blue staining. Mesenchymal stem cells could be isolated and cultured by the method of bone marrow adherent culture in vitro. However, bone marrow mesenchymal stem cells from rats at different ages exhibit decreased proliferation and differentiation abilities with the increase of age through determination of osteocalcin content.