体外长期培养的家猪骨髓间充质干细胞生物学特性变化

Change of the biological characteristics in long-term cultivated mesenchymal stem cells from porcine bone marrow

目的观察体外长期培养的家猪骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)生物学特性的变化。方法将第1代MSCs(早期)和第35代MSCs(晚期)分别进行细胞形态学观察和染色体核型分析;钙沉积和细胞增殖测定,以及采用实时定量PCR定量分析成骨分化及成脂分化相关基因表达。结果早期培养的MSCs细胞形态为较大的纺锤形,晚期为小的梭形。第35代MSCs细胞Ki-67染色强阳性,染色体数目增加。成骨诱导条件下早期MSCs茜素红S染色呈强阳性,晚期MSCs未见明显染色且OC基因表达明显下调(P<0.01);成脂诱导条件下早期MSCs油红O染色阳性,晚期MSCs未见明显染色且PPARγ2基因表达明显下调(P<0.01)。结论体外长期培养的家猪晚期MSCs不适合作为组织工程种子细胞。

Objective To observe the change of the biological characteristics in mesenchymal stem cells porcine bone marrow mesenchymal stem cells （MSCs） during long-term expansion in vitro. Methods Bone marrow from 6 landrace pigs, 3-monthold about 50 kg, was aspirated from the medullary cavity of the proximal tibia. The MSCs were isolated, and purified by Ficoll density gradient centrifugation combined with adherent culture method. Cell morphology from passage 1 and passage 35 were observed by microscope. Cell proliferation was tested by using Ki67 immunostaining. Karyotyping was also evaluated. Osteogenic and adipogenic differentiation were evaluated by histological assays and analysis of gene expression using real-time RT-PCR technology. Results MSCs showed a change in appearance, from the initial spindle shape to small contact shape. Ki67 staining from P35 showed strong positive and the number of chromosome was increased. In addition, muhilineage differentiation potency was lost. Osteogenic and adipogenic differentiation-related gene expression were markedly decreased （P〈0.01）.Conclusion Late stage MSCs are not suitable for tissue engineering due to their spontaneous transformation and progressively lose of multipotency.