摘要
目的探讨寡脱氧核苷酸(ODN)MT01对大鼠骨髓间充质干细胞(BMSCs)形态、增殖及成骨分化的影响。方法4周龄清洁级SD雄性大鼠5只,以全骨髓贴壁法培养SD大鼠BMSCs,通过观察不同代次的细胞形态、流式细胞仪检测表面相关标志物和成骨分化潜能,对所提取的干细胞进行鉴定。设立PBS组(对照组)和不同浓度(0.5、1.0、2.0、4.0μg/ml)ODN MT01组进行对照研究,每组实验重复3次。通过DAPI和鬼笔环肽荧光染色观察各组BMSCs细胞核及骨架的形态变化;通过CCK-8法检测不同时间各组BMSCs的增殖活性;成骨诱导培养基培养7 d和21 d,分别行碱性磷酸酶染色及其活性检测和茜素红染色鉴定各组BMSCs的成骨分化程度。组间两两比较采用t检验,多组间比较采用单因素方差分析,P<0.05为差异有统计学意义。结果随着代次增加,BMSCs纯度越来越高,第3代细胞整体呈鱼群样分布。流式细胞仪分析结果显示,细胞高表达CD29(99.8%)、CD44(96.1%),低表达CD11b(1.03%)、CD45(1.74%)。成骨诱导7 d后碱性磷酸酶染色及21 d后茜素红染色均为阳性。细胞核及骨架染色显示,ODN MT01浓度为4.0μg/ml时会导致BMSCs皱缩并降低伸展性。CCK-8结果显示,培养4 d时对照组吸光度值为0.446±0.018,1.0、2.0μg/ml ODN MT01组分别为0.505±0.019、0.528±0.014,与对照组比较差异有统计学意义(t=2.954、4.083,P=0.033、0.008),培养7 d时对照组吸光度值为0.514±0.027,1.0、2.0μg/ml ODN MT01组分别为0.607±0.007、0.636±0.023,与对照组比较差异有统计学意义(t=4.664、6.091,P=0.009、0.008),BMSCs增殖能力较对照组明显升高,而4.0μg/ml ODN MT01(0.427±0.013)对BMSCs增殖能力具有抑制作用(t=4.332,P=0.015);诱导BMSCs成骨分化过程中随着ODN MT01浓度的升高,蓝色团块和矿化结节均显著增加,培养7 d后的碱性磷酸酶活性升高趋势与ALP染色结果相类似,对照组碱性磷酸酶活性为1.207±0.023,0.5、1.0、2.0、4.0μg/ml ODN MT01�
Objective To investigate the effects of oligodeoxynucleotide(ODN)MT01 on the morphology,proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs).Methods The BMSCs of SD rat were isolated and cultured by direct adherence method.The extracted cells were identified by cell morphology of different generations,the expression of surface markers detected by flow cytometry and osteogenic differentiation potential.ODN MT01 group was set up in a gradient of concentrations(0.5,1.0,2.0,4.0μg/ml)and PBS group as control.Each group of experiments was repeated three times.The morphological changes of cell nucleus and cytoskeleton were fluorescent stained by DAPI and FITC-phalloidin,respectively.The proliferation activities of the BMSCs in different group were analyzed by CCK-8 assay at 1,4 and 7 d.The degrees of osteogenic differentiation of BMSCs in different group were assessed via alkaline phosphatase(ALP)staining,ALP activity assay and alizarin red S staining respectively on the 7th and 21st days after cultured in osteogenic induction medium.Statistical differences between two groups and among groups were analyzed by t-test and one-way ANOVA,respectively.Differences were regarded as statistically significant when a P value of less than 0.05.Results Flow cytometry showed that the BMSCs were positive for CD29(99.8%)and CD44(96.1%)while negative for CD11b(1.03%)and CD45(1.74%).ALP staining and alizarin red S staining were positive at different stages of osteogenesis induction confirmed that BMSCs was able to differentiate into the osteoblast.The nucleus and cytoskeleton staining showed that BMSCs were shrunk and the extensibility was reduced when the concentration of ODN MT01 was 4.0μg/ml.CCK-8 assay showed that the absorbance value of control group was 0.446±0.018,1.0μg/ml ODN MT01 was 0.505±0.019,2.0μg/ml ODN MT01 was 0.528±0.014 after cultured for 4 days.Compared with the control group,the difference is statistically significant(t=2.954,4.083,P=0.033,0.008).The absorbance value of
作者
陈宇
周平辉
管晶晶
梁梦想
张莉
毛颖基
Chen Yu;Zhou Pinghui;Guan Jingjing;Liang Mengxiang;Zhang Li;Mao Yingji(Department of Plastic Surgery,the First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China;College of Life Sciences,Bengbu Medical College,Bengbu 233030,China;Department of Orthopedics Surgery,the First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China;Anhui Key Laboratory of Tissue Transplantation,Bengbu Medical College,Bengbu 233030,China;Department of Laboratory Medicine,the First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China)
出处
《中华整形外科杂志》
CAS
CSCD
北大核心
2020年第5期560-567,共8页
Chinese Journal of Plastic Surgery
基金
国家自然科学基金青年科学基金项目(31700854)
安徽省教育厅自然科学重点项目(KJ2018A1011)
蚌埠医学院校科技发展基金项目(BYKF17118,BYKY1848ZD,BYKY18108)
蚌埠医学院研究生科研创新计划项目(Byycx1827)
安徽省大学生创新创业训练计划(201810367013,201810367028)。
关键词
寡脱氧核苷酸
骨髓间充质干细胞
形态学
细胞增殖
成骨分化
Oligodeoxynucleotides
Bone marrow mesenchymal stem cells
Morphology
Proliferation
Osteogenic differentiation
