肝素促进转化生长因子-β_3在体外诱导兔牙髓干细胞成骨向分化中的作用  

Role of heparin to promote transforming growth factor-β_3 in inducing rabbit dental pulp stem cells osteogenic differentiation in vitro

作　　者：麦麦提依明.哈力克 木合塔尔.霍加 刘晓文[1] 帕尔哈提.阿布都热合曼 

机构地区：[1]新疆维吾尔自治区人民医院口腔科,乌鲁木齐830001

出　　处：《中华实用诊断与治疗杂志》2016年第4期340-342,共3页Journal of Chinese Practical Diagnosis and Therapy

基　　金：国家自然科学基金(81560180)

摘　　要：目的探讨转化生长因子(transforming growth factor,TGF)-β_3联合肝素在体外诱导兔牙髓干细胞(dental pulp stem cells,DPSCs)成骨向分化中的能力。方法新西兰幼兔4只,采用酶解组织块法分离培养兔DPSCs,传代培养至第3代兔DPSCs,分为空白对照组、TGF-β_3组和TGF-β_3+肝素组;空白对照组正常培养,TGF-β_3组在细胞培养基中加入20μg/L TGF-β_3,TGF-β_3+肝素组在细胞培养基中同时加入20μg/L TGF-β_3和5u/mL肝素。采用碱性磷酸酶(alkailine phosphate,ALP)试剂盒检测各组兔DPSCs成骨向诱导后第7、14天细胞内ALP活性,细胞爬片采用免疫细胞化学法检测成骨细胞标记物RUNT相关转录因子2(runt related transcription factor 2,RUNX-2)和骨钙素(osteocalcin,OC)表达情况,采用茜素红染色观察矿化结节形成情况。结果兔DPSCs在诱导体系中生长状态良好;TGF-β_3+肝素组第7、14天ALP活性[(27.20±1.01)、(29.06±1.39)u/(mg·pro)]高于TGF-β_3组[(12.69±1.03)、(9.44±1.05)u/(mg·pro)]和对照组[(5.96±3.68)、(6.10±3.66)u/(mg·pro)](P<0.01),TGF-β_3组高于对照组(P<0.01);TGF-β_3组和TGF-β_3+肝素组成骨诱导第7天RUNX-2开始出现阳性表达,第14天OC有阳性表达,对照组均为阴性;第21天茜素红染色对照组为阴性,TGF-β_3组为阳性,TGF-β_3+肝素组为强阳性。结论肝素有促进TGF-β_3体外诱导兔DPSCs成骨向分化的能力。Objective To investigate the capacity of heparin to promote transforming growth factor-β_3（TGF-β_3）to induce rabbit dental pulp stem cells（DPSCs）osteogenic differentiation in vitro.Methods Rabbit DPSCs were isolated by tissue enzymatic digestion methods in 4 young New Zealand rabbits.The third-passage DPSCs cultured in vitro were divided into three groups,and incubated respectively with total medium（control group）,20μg/L TGF-β_3（TGF-β_3 group）,and 20μg/L TGF-β_3 in combination with 5u/mL heparin（TGF-β_3＋heparin group）.Alkailine phosphate（ALP）activity assay was adopted to detect ALP activity,immunocytochemistry assay was adopted to detect the expressions of runt related transcription factor-2（RUNX-2）and osteocalcin（OC）,and Alizarin red staining was adopted to observe the mineralized nodules.Results DPSCs grew well in inducing system.The values of ALP activity by day 7 and 14 after induction were significantly higher in TGF-β_3＋heparin group（（27.20±1.01）,（29.06±1.39）u/（mg·pro））than those in TGF-β_3 group（（12.69±1.03）,（9.44±1.05）u/（mg·pro））and control group（（5.96±3.68）,（6.10±3.66）u/（mg·pro））（P〈0.01）,and higher in TGF-β_3 group than those in control group（P〈0.01）.RUNX-2 showed positive by day 7,and OC was positive by day 14 after ostegenic induction in TGF-β_3 and TGF-β_3＋heparin groups,and both RUNX-2 and OC were negative in control group.Alizarin red staining was negative by day 21 in control group,was positive in TGF-β_3 group and was strong positive in TGF-β_3＋heparin group.Conclusion The additional application of heparin promotes the inductive capacity of TGF-β_3 on the osteogenic differentiation in vitro of DPSCs.

关 键 词：牙髓干细胞 转化生长因子-Β3 肝素 成骨向分化 兔 

分 类 号：R781[医药卫生—口腔医学]