兔骨髓间充质干细胞的分离培养及诱导分化

Isolated Culture and Induced Differentiation of The Rabbit Marrow Mesenchyme Stem Cells

目的:分离培养兔骨髓间充质干细胞并成骨诱导分化。方法:从兔骨髓中分离培养原代MSCs并进行传代培养,绘制生长曲线。取第3代细胞进行成骨诱导分化,进行Vonkossa染色,碱性磷酸酶染色,碱性磷酸酶(ALP)活性测定,总蛋白检测,提取细胞总RNA及RT-PCR法检测Collagen I,osteocalcin及osteopontin基因表达情况。结果:MSCs贴壁生长,呈放射样、爆花样或漩涡样,可传20代以上。未经成骨诱导的MSCs不发生矿化沉积,而经成骨诱导分化后,细胞可发生显著的矿质沉积。未经成骨诱导的MSCs可微量表达CollagenI,但不表达osteocalcin和osteopontin,而经成骨诱导分化后,细胞表达CollagenI增强,并出现osteocalcin和osteopontin等成骨标志物的表达。结论:本实验成功分离培养MSCs并成骨诱导分化。

Objective：isolated culture the rabbit marrow mesenchyme stem cells（MSCs） and induced differentiated ossificationly.Methods：isolated culture and subculturing the origin MSCs from the rabbit marrow, draw the cells’ growth curve. induced differentiated the third generation cells ossificationly, Vonkossa staining the cells, alkaline phosphatase staining the cells, detect the alkaline phosphatase activity, detect the cells’ total protein, abstract cells’ total RNA and detect Collagen I, osteocalcin and osteopontin’s gene expression using RT-PCR method.Results：MSCs growth adherencly with radiational, explosive or whirlpoolly shape. The cells could passage above 20 generation. The MSCs unossificationally induced show no mineralized deposition, while being ossificationally induced, the MSCs developped noticeable mineralized deposition. The MSCs unossificationally induced gleamly express Collagen I, but don’t express osteocalcin and osteopontin, while being ossificationally induced, the MSCs strengthenly express Collagen I, and show the expression of osteocalcin and osteopontin genes.Conclusion：The experiment successfully isolated culture the rabbit MSCs and induced differentiated ossificationly.