摘要
目的 探讨适宜培养乳鼠原代成骨细胞的培养基配比及培养时间,为体外成骨细胞原代培养提供改良实验方案。方法 用间断酶消化法消化CD1乳鼠颅骨提取原代成骨细胞,差速离心获得纯化成骨细胞。制定诱导时间、胎牛血清、β-甘油磷酸钠盐、地塞米松浓度梯度实验,用Western blot与免疫荧光测定成骨细胞成熟标志物,通过碱性磷酸酶、茜素红染色、超微结构鉴定其成骨活性。结果 (1)间断酶消化法可从乳鼠颅骨中获得原代细胞进行增殖及传代扩增,细胞具有典型成骨细胞形态学和生物学活性。(2)成骨细胞成熟标志物表达与诱导培养时间、胎牛血清浓度成平行关系,使用10%血清培养14 d即可获得成熟成骨细胞。(3)分别在不同浓度含β-甘油磷酸钠盐及地塞米松培养液中诱导原代成骨细胞分化,于10 mmol/L β-甘油磷酸钠盐及5 nmol/L地塞米松培养条件下成骨细胞成熟标志物表达较高(P<0.01),碱性磷酸酶及茜素红染色明显,成骨活性良好。结论 CD1乳鼠颅骨提取的原代成骨细胞在含10%胎牛血清、10 mmol/L β-甘油磷酸钠盐溶液及5 nmol/L地塞米松的诱导培养基中培养,于14 d即有良好的成骨活性,适用于做体外实验研究。
Objective To develop a suitable medium and optimize culture time for the primary osteoblast culture from suckling mouse,so to provide an improved experimental protocol for primary osteoblast culture in vitro.Methods Primary osteoblasts were collected from skull of CD1 suckling mouse by interrupted enzyme digestion.The purified osteoblasts were harvested by differential centrifugation.The incubation time,concentration of fetal bovine serum(FBS),β-glycerophosphate sodium and dexamethasone were tested and optimized.The change of osteoblast maturation marker was examined by Western blot(WB)and immunofluorescence staining(IF).The osteogenic activity was determined by alkaline phosphatase staining,alizarin red staining and ultrastructure.Results Primary osteoblast were obtained from sucleling mouse skull bone by interrupted enzyme digestion for proliferation and transgenerational expansion.The expression of osteoblast maturation markers was parallel to the time of induction culture and the concentration of FBS.Mature osteoblasts were obtained by culturing the cells with 10%FBS for 14 days.The differentiation of primary osteoblasts was induced by different concentrations ofβ-glycerophosphate and dexamethasone.The results showed that the expression of osteoblast maturation markers was higher under the culture conditions of 10 mmol/Lβ-glycerophosphate and 5 nmol/L dexamethasone(P<0.01),and the staining of alkaline phosphatase and alizarin red was obvious,and the osteogenic activity was better too.Conclusions Primary osteoblasts isolated from the skull of suckling CD1 mice cultured in induction medium containing 10%fetal bovine serum,10 mmol/Lβ-glycerophosphate sodium and 5 nmol/L dexamethasone for 14 days show good osteogenic activity and are suitable for in vitro experimental studies.
作者
王左钰
周阳
杨俊伟
WANG Zuoyu;ZHOU Yang;YANG Junwei(Center for Kidney Disease,the Second Affiliated Hospital of Nanjing Medical University,Nanjing 210003,China)
出处
《基础医学与临床》
2024年第2期159-166,共8页
Basic and Clinical Medicine
基金
国家自然科学基金(82270760)。
关键词
乳鼠
成骨细胞
原代培养
suckling mouse
osteoblasts
primary culture
