增龄因素对牙源干细胞生物学特性的影响

Effects of aging factors on biological characteristics of dental stem cells

背景:牙源干细胞在再生医学与组织工程等领域的研究不断深入,为牙相关组织修复及全身疾病治疗带来了希望。但目前对不同年龄区间牙源干细胞生物学特性上的差异缺少系统的分析。目的:探讨脐血源血小板裂解液培养人乳牙、恒牙牙髓干细胞的生物学特性,为人血小板裂解液代替胎牛血清提供可靠依据。方法:取出乳牙、少年恒牙及成年恒牙牙髓组织,在含体积分数为10%胎牛血清或5%,10%,15%人血小板裂解液的DMEM/F-12培养基中培养,采用细胞计数法检测4组细胞增殖情况,选取最佳人血小板裂解液浓度进行后续实验。在最佳人血小板裂解液浓度条件下,体外培养乳牙、少年恒牙及成年恒牙牙髓干细胞,显微镜下观察细胞生长状态,流式细胞术检测细胞表面特异性抗原,细胞计数法和CCK-8法检测细胞增殖能力,三系分化实验观察细胞体外分化能力。结果与结论:①10%人血小板裂解液组的细胞增殖速率最高;②各组牙髓组织块周围均有梭形成纤维状细胞生长并扩增,乳牙与少年恒牙细胞形态无明显差异;与同代次乳牙和少年恒牙细胞相比,成年恒牙细胞体积偏大;③流式细胞术检测结果显示乳牙、少年恒牙及成年恒牙牙髓干细胞均符合间充质来源干细胞的表型特征;④成年恒牙牙髓干细胞增殖能力明显低于乳牙及少年恒牙牙髓干细胞(P<0.01);⑤成年恒牙牙髓干细胞中成骨相关基因碱性磷酸酶、骨形态发生蛋白2 mRNA表达,成脂相关基因脂蛋白脂肪酶、过氧化物酶体增殖物激活受体γ2 mRNA表达,成软骨相关基因Ⅱ型胶原α1、软骨寡聚基质蛋白mRNA表达均显著低于乳牙及少年恒牙牙髓干细胞(P<0.01);⑥结果表明,人乳牙与少年恒牙牙髓干细胞相较于成年恒牙牙髓干细胞具有更强的增殖能力与多向分化潜能,更适用于临床研究和疾病治疗。

BACKGROUND:The research of dental stem cells in the fields of regenerative medicine and tissue engineering has been deepening,bringing hope for the repair of tooth-related tissues and the treatment of systemic diseases.However,there is a lack of systematic research and analysis on the biological characteristics of dental stem cells in different age groups.OBJECTIVE:To explore the biological characteristics of the human deciduous tooth and permanent tooth pulp stem cells cultured in umbilical cord blood platelet lysate to provide a reliable basis for human platelet lysates to replace fetal bovine serum.METHODS:The pulp tissues of deciduous teeth,juvenile permanent teeth and adult permanent teeth were taken out and cultured in DMEM/F-12 medium supplemented with 10%fetal bovine serum or different concentrations(5%,10%and 15%)of human platelet lysates.Cell proliferation in the four groups was detected by cytometry.The optimal concentration of human platelet lysates was selected for subsequent experiments.Under the optimal concentration of human platelet lysates,human deciduous tooth and juvenile and adult permanent tooth pulp stem cells were cultured in vitro.The cell growth status was observed under the microscope.The specific antigen on the cell surface was detected by flow cytometry.The cell proliferation ability was tested by the cell counting method and CCK-8 assay.The cell differentiation ability in vitro was observed by a three-line differentiation assay.RESULTS AND CONCLUSION:(1)The cell proliferation rate of the 10%human platelet lysate group was the highest.(2)In all three groups,fusiform fibrous cells grew and expanded from around the tissue block.There was no significant difference between deciduous teeth and juvenile permanent tooth cells,but the adult permanent tooth cells were larger than the deciduous and juvenile permanent tooth cells of the same generation.(3)The results of flow cytometry showed that deciduous teeth,juvenile permanent teeth and adult permanent teeth conformed to the phenotypic characteristics of mesenchymal stem cells.(4)The proliferative capacity of adult permanent dental pulp stem cells was significantly lower than those of deciduous teeth and juvenile permanent dental pulp stem cells(P<0.01).(5)mRNA expressions of osteoblast-related genes alkaline phosphatase and bone morphogenetic protein 2,lipoprotein lipase and peroxisome proliferator-activated receptorγ2,mRNA expressions of chondroblast related gene type II collagenα1 and cartilage oligomeric matrix protein in adult pulp stem cells of permanent teeth were significantly lower than those of deciduous teeth and juvenile permanent teeth pulp stem cells(P<0.01).(6)Compared with adult dental pulp stem cells,human deciduous teeth and juvenile permanent teeth dental pulp stem cells have the stronger proliferative capacity and multidirectional differentiation potential,and are more suitable for clinical research and disease treatment.