摘要
目的 建立永生化大鼠成骨细胞系ROB-1并探讨氟对其DNA的损伤。方法 用酶消化法原代分离SD胎鼠颅骨成骨细胞,用Lipofect AMINE作载体对其转染SV40大T基因,并经PCR法鉴定大T基因整合情况。采用碱性磷酸酶染色法鉴定转染细胞系中成骨细胞的纯度;采用单细胞凝胶电泳(SCGE)方法检测2.0～3.0mmol/L氟化钠染毒24h对成骨细胞DNA损伤情况。结果 转染后成骨细胞的基因组中整合有SV40T基因,碱性磷酸酶染色阳性细胞占95％以上;单细胞凝胶电泳发现NaF可不同程度地诱导细胞DNA链断裂,与溶剂对照组相比,各剂量组DNA断裂细胞百分率及其彗尾长度均有统计学意义的增加(P<0.05)。结论 建立了永生化大鼠成骨细胞系ROB-1;氟对成骨细胞DNA有损伤作用。
Objective To establish the immortalized rat osteoblast cell line ROB - 1 and detect its DNA damage induced by fluoride.Methods Primary calvarial osteoblasts from embryo rat (SD) were isolated by enzymic digestion. Semian virus 40 early region large T gene and lipofect AMINE were co - transfected into the primary osteoblasts. PCR was used to detect the homologous integration of SV40 T gene into the primary osteoblasts genome. The purity of osteoblasts was identified by alkaline phosphatase staining. Single - cell gel electrophoresis was employed to detect DNA damage of ROB - 1 after induction by excessive fluoride (NaF: 2.0, 2.5 and 3.0 mmol/L, 24 h ). Results Homologous integration of SV40T gene into RAE - 1 cell genome was positively detected by PCR. More than 95% cells were proved positively by alkaline phosphatase staining. Single - cell gel electrophoresis showed that NaF could induce osteoblast DNA strands breakage in a dose - dependent manner. Compared with control group, the percentage of DNA - broken cells and the DNA tail length of NaF treated groups were significantly increased ( P < 0.05 ). Conclusion Immortalized rat osteoblast cell line ROB - 1 was established successfully. Excessive fluoride could cause DNA damage of osteoblasts.
出处
《中国预防医学杂志》
CAS
2002年第3期191-194,共4页
Chinese Preventive Medicine
基金
国家重点自然科学基金(项目号:39730390)
