CAJ | 학술논문

目的观察共培养条件下电磁场干预对大鼠成骨细胞及骨髓间充质干细胞(MSCs)成骨定向分化的影响,并探讨电磁场促进成骨分化的相关机制.方法体外分离培养SD大鼠BMSCs及成骨细胞,将第三代成骨细胞与BMSCs通过transwell培养小室建立共培养系统.采用随机数字表法将共培养细胞分为共培养组及共培养暴磁组,另随机选择单细胞培养的BMSCs及成骨细胞纳入单细胞培养组.共培养暴磁组细胞每日给予电磁场刺激4h.于实验进行14 d后随机提取各组细胞总RNA,采用荧光定量PCR技术检测各组细胞Runx2、Sp7、碱性磷酸酶、Ⅰ型胶原、骨形态形成蛋白-2及骨钙素基因表达情况,并选用茜素红染色法检测各组细胞矿化钙结节形成情况.结果单细胞培养模式下BMSCs及成骨细胞其各项成骨相关基因表达水平均较低,而共培养模式下BMSCs及成骨细胞其各项成骨相关基因表达均呈现不同程度增强,并且以共培养暴磁组成骨细胞上述指标增强幅度较显著.通过茜素红染色发现,与单纯共培养组比较,共培养暴磁组细胞矿化钙结节数量明显增多.结论低频电磁场干预可显著促进共培养条件下BMSCs及成骨细胞成骨定向分化,其可能机制为电磁场刺激能促进骨形态形成蛋白-2表达,使其通过自分泌或旁分泌方式介导细胞间信号转导,从而诱导BMSCs向成骨细胞分化及成骨细胞进一步成熟分化.
목적 관찰공배양조건하전자장간예대대서성골세포급골수간충질간세포(MSCs)성골정향분화적영향,병탐토전자장촉진성골분화적상관궤제.방법 체외분리배양SD대서BMSCs급성골세포,장제삼대성골세포여BMSCs통과transwell배양소실건립공배양계통.채용수궤수자표법장공배양세포분위공배양조급공배양폭자조,령수궤선택단세포배양적BMSCs급성골세포납입단세포배양조.공배양폭자조세포매일급여전자장자격4h.우실험진행14 d후수궤제취각조세포총RNA,채용형광정량PCR기술검측각조세포Runx2、Sp7、감성린산매、Ⅰ형효원、골형태형성단백-2급골개소기인표체정황,병선용천소홍염색법검측각조세포광화개결절형성정황.결과 단세포배양모식하BMSCs급성골세포기각항성골상관기인표체수평균교저,이공배양모식하BMSCs급성골세포기각항성골상관기인표체균정현불동정도증강,병차이공배양폭자조성골세포상술지표증강폭도교현저.통과천소홍염색발현,여단순공배양조비교,공배양폭자조세포광화개결절수량명현증다.결론 저빈전자장간예가현저촉진공배양조건하BMSCs급성골세포성골정향분화,기가능궤제위전자장자격능촉진골형태형성단백-2표체,사기통과자분비혹방분비방식개도세포간신호전도,종이유도BMSCs향성골세포분화급성골세포진일보성숙분화.
Objective To explore the effects of electromagnetic fields (EMFs) on osteogenesis during co-culture of bone mesenchymal stem cells (BMSCs) with osteoblasts in rats.Methods BMSCs and osteoblasts were isolated from Sprague-Dawley rats and cultured.Sub-cultured osteoblasts and BMSCs were seeded in transwell cell-culture-chamber polyester inserts to establish the co-culture system.The co-cultures were then randomly divided into a normal co-culture group and a group exposed to an EMF.Single-cultured BMSCs and osteoblasts were set as a single culture group.The EMF group was exposed to an EMF for 4 hours per day.On the 14th day,cell culture plates or inserts were randomly selected for total RNA extraction and measurement of the mRNA expression levels of Runt-related transcription factor 2,transcription factor 7,alkaline phosphatase,collagen type Ⅰ,bone morphogenetic protein 2 (BMP-2) and bone gamma-carboxyglutamate protein (Osteocalcin gene,OC gene) using real-time PCR assays.Cell culture dishes or inserts were also randomly chosen for Alizarin red staining to detect mineralized nodules.Results The level of osteogenic gene expression in single-cultured BMSCs and osteoblasts was low,while it was much higher in the co-culture group.The level of gene expression in the EMF-exposed and co-cultured group was even higher.Alizarin red staining also showed that calcium mineralized modules had increased in the stimulated,co-cultured system compared with the unstimulated,co-cultured cells.Conclusion EMF exposure can promote osteogenic differentiation of BMSCs and osteoblasts when they are co-cultured.BMP2-mediated cellular interaction might play an important role in osteogenic differentiation induced by EMF exposure.