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目的探讨(±)Bay K8644对衰老的大鼠(24 m)来源的骨髓间充质干细胞(Aging bone marrow stromal cells,Aging-BMSCs)的骨向分化能力的影响。方法通过全骨髓贴壁培养法获得大鼠BMSCs,使用β-半乳糖苷酶染色方法鉴定细胞的衰老状态,随后应用(±)Bay K8644对细胞进行干预。以干性标志物Nanog为靶点,通过实时定量PCR筛选出(±)Bay K8644作用的最佳浓度,随后对细胞进行成骨分化诱导,并检测细胞在药物干预下的骨向分化能力的改变。在诱导第14天后对成骨分化标志酶碱性磷酸酶进行染色及活性测定、第21天后进行茜素红染色。结果(±)Bay K8644明显的降低了Aging-BMSCs细胞内β-半乳糖苷酶的含量(P<0.05),同时干细胞的干性标记物Nanog的表达明显提升(P<0.05)。对细胞进行成骨分化诱导后,成骨分化标志基因表达上调,ALP、茜素红染色的结果提示实验组骨向分化水平明显较对照组提升(P<0.05)。结论 (±)Bay K8644能够明显改善衰老骨髓间充质干细胞的衰老状态,并使其骨向分化的能力得到提升。
Objective To investigate the effect of (±) Bay K8644 on the osteogenic differentiation of Aging bone marrow stromal cells (Aging-BMSCs) from aged rats (24 m). Methods Rat BMSCs were obtained by whole bone marrow adherent culture. The senescence status of the cells was identified by β-galactosidase staining, and then the cells were treated with (±) Bay K8644. The optimal concentration of (±) Bay K8644 was screened out by real-time quantitative PCR using Nanog as a target. Subsequently, the cells were induced to differentiate into osteoblasts and the differentiation of cells into osteoblasts under drug intervention . After 14 days of induction, the osteoblast differentiation-inducing enzyme alkaline phosphatase was stained and assayed, and after 21 days, alizarin red staining was performed. Results (±) Bay K8644 significantly decreased the content of β-galactosidase in Aging-BMSCs (P <0.05), and the expression of stem cell marker Nanog was significantly increased (P <0.05). After induced by osteogenic differentiation, the gene expression of osteogenic differentiation marker was up-regulated. The results of ALP and alizarin red staining suggested that the bone differentiation level in experimental group was significantly higher than that in control group (P <0.05). Conclusion (±) Bay K8644 can significantly improve the aging status of aging mesenchymal stem cells and enhance their osteogenic differentiation.