摘要
目的探讨转化生长因子-β_(3)(transforming growth factor-β_(3),TGF-β_(3))体外诱导人牙髓干细胞(human dental pulp stem cells,hDPSCs)成骨向分化的效果及可能机制。方法采用免疫荧光技术观察原代hDPSCs基质细胞抗原STRO-1的免疫荧光染色情况,应用流式细胞仪检测原代hDPSCs细胞CD90、CD73、CD105表达情况,鉴别是否为hDPSCs。应用倒置显微镜观察培养第1~7天hDPSCs形态变化。原代hDPSCs传代培养至第3代对数生长期,随机分为空白组、实验组、对照组,空白组继续在常规培养基中培养,实验组在常规培养基中加入80μg/L TGF-β_(3)后培养,对照组在常规培养基中加入10 mmol/Lβ-甘油磷酸盐、100 nmol/L地塞米松、50 mg/L抗坏血酸后培养。培养第7天,采用实时荧光定量PCR法检测3组细胞TGF-βⅠ型受体(TGF-βreceptor typeⅠ,TGF-βRⅠ)、Smad2、Smad3、Smad4 mRNA相对表达量,采用Western blot法检测3组细胞骨钙素(osteocalcin,OCN)、Runx-2、TGF-βRⅠ、Smad2/Smad3、p-Smad2、p-Smad3、Smad4蛋白相对表达量;培养第14、21天,采用茜素红染色观察3组钙化结节情况。结果原代hDPSCs基质细胞抗原STRO-1免疫荧光染色呈阳性,hDPSCs细胞表面CD90、CD73、CD105阳性表达率分别为93.4%、98.2%、89.1%;鉴定原代细胞为hDPSCs。第3代hDPSCs培养第1~3天,细胞多呈不规则多角形、梭形或三角形;培养第4~7天,细胞逐渐呈长条状梭形。培养第7天,实验组细胞TGF-βRⅠ、Smad2、Smad3、Smad4 mRNA相对表达量(1.69±0.25、1.84±0.15、1.73±0.29、1.79±0.25)均高于空白组(1.02±0.30、1.02±0.30、1.00±0.25、1.01±0.26)(P<0.05),Smad2、Smad4 mRNA相对表达量均高于对照组(1.41±0.11、1.33±0.12)(P<0.05),TGF-βRⅠ、Smad3 mRNA相对表达量与对照组(1.50±0.12、1.43±0.24)比较差异均无统计学意义(P>0.05);对照组细胞TGF-βRⅠmRNA相对表达量高于空白组(P<0.05),Smad2、Smad3、Smad4 mRNA相对表达量与空白组比较差异均无统计学意义(P>0.05
Objective To investigate the effect of transforming growth factor-β_(3)(TGF-β_(3))on inducing the osteogenic transformation of human dental pulp stem cells(hDPSCs)in vitro and its potential mechanism.Methods Immunofluorescence technique was used to observe the immunofluorescence staining of stromal cell antigen STRO-1 of primary hDPSCs.Flow cytometry was used to detect the expressions of CD90,CD73 and CD105 in primary hDPSCs to identify whether they were hDPSCs.The morphological changes of cells were observed by inverted microscope from day 1 to 7of culture.The primary hDPSCs were cultured to the third generation in logarithmic growth phase,which were randomly divided into blank group,experimental group and control group.Blank group continued to be cultured normally in conventional medium,experimental group was cultured after adding 80μg/L TGF-β_(3)into conventional medium,and control group was cultured with 10 mmoLβ-glycerophosphate,100nmol/L dexamethasone and 50mg/L ascorbic acid in conventional medium.After 7days of culture,the relative expressions of TGF-βreceptor typeⅠ(TGF-βRⅠ),Smad2,Smad3and Smad4 mRNAs were detected by real-time fluorescence quantitative PCR.The relative expressions of osteocalcin(OCN),Runx-2,TGF-βRⅠ,Smad2/Smad3,p-Smad2,p-Smad3and Smad4proteins were detected by Western blot.On the 14th and 21st days of culture,alizarin red staining was used to observe the calcified nodules in three groups.Results Immunofluorescence staining of primary hDPSCs stromal cell antigen STRO-1showed positive.The positive rates of CD90,CD73and CD105 were 93.4%,98.2%and 89.1%,respectively.The primary cells were identified as hDPSCs.The cell morphology was irregular polygon,spindle or triangle from day 1to 3 of culture,and gradually changed to elongated spindle from day 4to 7of culture.On the 7th day of culture,the relative expressions of TGF-βRⅠ,Smad2,Smad3 and Smad4 mRNAs were higher in experimental group(1.69±0.25,1.84±0.15,1.73±0.29,1.79±0.25)than those in blank group(1.02±0.30,1.02±0.30,1.
作者
孙帅
李军
陈炼
邵静宇
姚清婷
张晓莉
SUN Shuai;LI Jun;CHEN Lian;SHAO Jing-yu;YAO Qing-ting;ZHANG Xiao-li(Department of General Stomatology,Stomatological Hospital of Xuzhou,Xuzhou,Jiangsu 221000,China;Department of Oral and Maxillofacial Surgery,People's Hospital of Xinjiang Uygur Autonomous Region,Urumqi,Xinjiang Uygur Autonomous Region 830001,China;Department of Oral and Maxillofacial Surgery,Three Gorges Hospital of Chongqing University,Chongqing 404000,China;Department of Stomatology,People's Hospital of Xinjiang Uygur Autonomous Region,Urumqi,Xinjiang Uygur Autonomous Region 830001,China)
出处
《中华实用诊断与治疗杂志》
2022年第4期352-357,共6页
Journal of Chinese Practical Diagnosis and Therapy
基金
新疆维吾尔自治区自然科学基金(2017D01C143)。
