衰老原代小鼠成骨细胞模型的建立与评价

Establishment and Evaluation of Osteoblast Model in Aging Primary Mice

目的提取小鼠颅骨原代成骨细胞,并建立成骨细胞衰老模型。方法取5只新出生5~7 d的昆明乳鼠颅骨,采取酶消化法与组织块培育法获取原代成骨细胞,经鉴定后,采用D-半乳糖诱导成骨细胞衰老,行β-半乳糖苷酶衰老染色、RT-PCR法分析ALP,BMP-2的表达。结果光学显微镜下观察细胞呈三角形,长梭形,不规则多边形,碱性磷酸酶染色呈阳性且阳性率为90%。经D-半乳糖处理后,成骨细胞β-半乳糖苷酶衰老染色阳性细胞数较对照组阳性细胞数显著增多(P<0.01),RT-PCR结果表明衰老成骨细胞中mRNAALP,BMP-2的表达较对照组均显著降低(P<0.05)。结论该研究采取D-半乳糖诱导法成功建立了小鼠原代成骨细胞衰老模型。

Objective To extract primary osteoblasts from mouse skull and establish an osteogenic aging model. Methods Five newborn rabbits of 5 to 7 days old Kunming suckling rats were used to obtain primary osteoblasts by enzymatic digestion and tissue block culture. After identification, D-galactose was used to induce osteoblast senescence, and β- Galectinase senescence staining, RT-PCR analysis of ALP, BMP-2 expression. Results Under light microscope, the cells were triangular, long fusiform and irregular polygon. The alkaline phosphatase staining was positive and the positive rate was 90%. After treatment with D-galactose, the number of positive cells staining positively for β-galactosidase in osteoblasts was significantly higher than that in the control group (P<0.01), RT-PCR results showed that senescent osteoblasts expression of mRNAALP and BMP-2 was significantly lower than that of the control group (P<0.05). Conclusion The mouse model of primary osteoblast senescence was successfully established by D -galactose induction method.