摘要
背景:现阶段骨形态发生蛋白2和碱性成纤维生长因子2对骨髓间充质干细胞膜片增殖、成骨分化的影响和作用机制还尚未可知,如何将生长因子与组织工程细胞膜片技术相整合,最终将其用于骨缺损修复具有重要意义。目的:探讨单独及联合应用骨形态发生蛋白2和碱性成纤维生长因子2对骨髓间充质干细胞膜片增殖和成骨分化的影响。方法:体外分离培养鉴定SD大鼠骨髓间充质干细胞并构建细胞膜片,选用不同质量浓度的骨形态发生蛋白2和碱性成纤维生长因子2单独及联合诱导骨髓间充质干细胞膜片,CCK-8法结合碱性磷酸酶活性检测确定2种因子促进膜片增殖和成骨分化的最佳有效质量浓度;然后对骨髓间充质干细胞膜片进行成骨诱导,通过大体及显微镜观察、Vonkossa染色、茜素红染色、RT-PCR检测相关成骨标志物来评估诱导效果。结果与结论:单独应用骨形态发生蛋白2可增强骨髓间充质干细胞膜片的碱性磷酸酶活性,最佳质量浓度为100μg/L(P<0.001),单独应用碱性成纤维生长因子2能加速骨髓间充质干细胞膜片的增殖,最佳质量浓度为20μg/L(P<0.001),而联合应用既可以促进膜片增殖又能提高其碱性磷酸酶活性(P<0.001);经成骨诱导后,4组膜片在形态学上无明显差异,均能诱导骨髓间充质干细胞膜片的成骨分化,其中联合组钙结节最明显(P<0.001),可显著促进膜片晚期成骨分化并抑制其早期成骨分化,具有明显的协同促进作用(P<0.001)。结果表明,骨形态发生蛋白2和碱性成纤维生长因子2联合应用时具有协同作用,既可以促进骨髓间充质干细胞膜片增殖,又能显著增强其成骨诱导能力。
BACKGROUND:The effects and mechanisms of bone morphogenetic protein 2 and basic fibroblast growth factor 2 on the proliferation and osteogenic differentiation of bone mesenchymal stem cells still remain unknown.How to combine the growth factors with tissue-engineered cell patch clamp techniques is of great significance for bone defect repair.OBJECTIVE:To explore the effects of bone morphogenetic protein 2 and basic fibroblast growth factor 2 applied alone or in combination on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cell sheet.METHODS:Bone marrow mesenchymal stem cells from Sprague-Dawley rats were isolated,cultured and identified in vitro to construct cell sheet.Bone morphogenetic protein 2 and basic fibroblast growth factor 2 at different concentrations were individually or jointly used to induce bone marrow mesenchymal stem cell sheet.The cell counting kit-8 assay combined with alkaline phosphatase activity assay was used to determine the optimal concentration of the two factors in promoting cell proliferation and osteogenic differentiation.Osteogenic induction of bone marrow mesenchymal stem cell sheet was assessed by gross and microscopic observations,Vonkossa staining,alizarin red staining,and RT-PCR detection.RESULTS AND CONCLUSION:The single application of bone morphogenetic protein 2 enhanced the alkaline phosphatase activity of the bone marrow mesenchymal stem cell sheet,and the optimal concentration was 100μg/L(P<0.001).The single application of basic fibroblast growth factor 2 accelerated the proliferation of bone marrow mesenchymal stem cell sheet,and the optimal concentration was 20μg/L(P<0.001).Their combination facilitated the proliferation of the cell sheet,and boosted the alkaline phosphatase activities(P<0.001).The four groups of cell sheet showed no significant morphological difference,and the osteogenic differentiation of the bone marrow mesenchymal stem cell sheet could all be induced through the osteogenic induction.Calcium nodules were most signifi
作者
张文静
王佳
田梦婷
韩祥祯
周琦琪
何惠宇
Zhang Wenjing;Wang Jia;Tian Mengting;Han Xiangzhen;Zhou Qiqi;He Huiyu(Department of Prosthodontics,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,Xinjiang Uygur Autonomous Region,China;School of Stomatology,Xinjiang Medical University,Urumqi 830054,Xinjiang Uygur Autonomous Region,China)
出处
《中国组织工程研究》
CAS
北大核心
2020年第1期65-71,共7页
Chinese Journal of Tissue Engineering Research
基金
新疆维吾尔自治区科技支疆项目(2018E02060),项目负责人:何惠宇~~
关键词
骨形态发生蛋白2
成纤维细胞生长因子2
细胞膜片技术
细胞增殖
成骨分化
bone morphogenetic protein 2
fibroblast growth factor 2
cell patch clamp technique
cell proliferation
osteogenic differentiation
