CAJ | 학술논문

目的观察碱性成纤维生长因子(bFGF)、表皮生长因子(EGF)和血小板衍生因子(PDGF-BB)对小鼠骨髓间充质干细胞(BMSCs)体外增殖和成骨分化的影响.方法采用贴壁培养纯化小鼠BMSCs,培养液(DMEM/F12,10％胎牛血清,青、链霉素)中添加(实验组)或者不添加(对照组)细胞因子bFGF、EGF和PDGF-BB(浓度均为10μg/L)进行培养,取传至第3代的细胞进行成骨分化,成骨诱导液:1×10-8 mol/L地塞米松、50 μmol/L抗坏血酸、10 mmol/L β-甘油磷酸钠.流式细胞仪检测细胞表面标记抗原CD45和CD90的表达；SYBgreen荧光定量聚合酶链反应(PCR)扩增检测成骨细胞特异性基因骨钙素和Ⅰ型胶原的表达；碱性磷酸酶染色和茜素红染色检测成骨细胞的分化.结果初始接种后,对照组的小鼠BMSCs贴壁较快,而传代后实验组的小鼠BMSCs增殖较快.对照组和实验组的小鼠BMSCs经传代纯化后CD45、CD90的阳性表达率分别为(27.34±0.69)％、(41.93 ±0.34)％和(0.48±0.12)％、(82.78 ±0.25)％,实验组的小鼠BMSCs在传代过程中较少观察到细胞的老化现象,细胞的纯化程度较高,而且其向成骨分化的效率明显高于对照组细胞.结论联合使用细胞因子bFGF、EGF和PDGF-BB可促进小鼠间BMSCs的体外增殖和干性的维持,并且有利于其向成骨细胞的分化.
목적 관찰감성성섬유생장인자(bFGF)、표피생장인자(EGF)화혈소판연생인자(PDGF-BB)대소서골수간충질간세포(BMSCs)체외증식화성골분화적영향.방법 채용첩벽배양순화소서BMSCs,배양액(DMEM/F12,10％태우혈청,청、련매소)중첨가(실험조)혹자불첨가(대조조)세포인자bFGF、EGF화PDGF-BB(농도균위10μg/L)진행배양,취전지제3대적세포진행성골분화,성골유도액:1×10-8 mol/L지새미송、50 μmol/L항배혈산、10 mmol/L β-감유린산납.류식세포의검측세포표면표기항원CD45화CD90적표체；SYBgreen형광정량취합매련반응(PCR)확증검측성골세포특이성기인골개소화Ⅰ형효원적표체；감성린산매염색화천소홍염색검측성골세포적분화.결과 초시접충후,대조조적소서BMSCs첩벽교쾌,이전대후실험조적소서BMSCs증식교쾌.대조조화실험조적소서BMSCs경전대순화후CD45、CD90적양성표체솔분별위(27.34±0.69)％、(41.93 ±0.34)％화(0.48±0.12)％、(82.78 ±0.25)％,실험조적소서BMSCs재전대과정중교소관찰도세포적노화현상,세포적순화정도교고,이차기향성골분화적효솔명현고우대조조세포.결론 연합사용세포인자bFGF、EGF화PDGF-BB가촉진소서간BMSCs적체외증식화간성적유지,병차유리우기향성골세포적분화.
Objective To investigate the effects of cytokine factors basic fibroblast growth factor (bFGF),epidermal growth factor (EGF) and platelet derived growth factor-BB (PDGF-BB) on prolification and osteogenesis of murine mesenchymal stem cells obtained from bone marrow (BMSCs).Methods Cell adhesion was used to purify the BMSCs in the presense or absence of bFGF,EGF and PDGF-BB (10 ag/L)in culture medium composed of DMEM/F12,10％ FBS,and penicillin/streptomycin (100 U/ml and 100 mg/L,respectively).Cells at the third passages were used for subsequent experiments.Flow cytometry was applied to detect the expression of cell-specific surface antigens CD45 and CD90.Osteogenic differentiation of BMSCs was measured by osteoblast-specific genes expression of collagen Ⅰ and osteocalcin,alkaline phosphatase assays and deposition of calcium mineralization.Results Cells in the absence of cytokines showed much faster adhesion rate than in the presence of cytokines after initial culture,but slower proliferative ability after passage,and displayed more senescense form.The expression of CD45 and CD90 in cells in the presence or absence of cytokines was [(0.48 ± 0.12)％,(82.78 ± 0.25)％] and [(27.34 ± 0.69)％,(41.93 ± 0.34)％],respectively.BMSCs in the presence of cytokines exhibited higher expression of osteoblast-specific genes,and better formation of calcium mineralization than control groups,indicating cells in the presence of cytokines possessed high potency for osteogenic differentiation.Conclusion Combination of three cytokines could improve the proliferation of mesenchymal stem cells in vitro and facilitate osteogenic differentiation.