摘要
目的:探讨长链非编码RNA(lncRNA)生长停滞特异性转录本5(GAS5)靶向miR-103对人牙髓干细胞(hDPSCs)向成牙骨质分化的影响。方法:从成人第三磨牙中分离培养hDPSCs,倒置光学显微镜观察P3代hDPSCs形态,流式细胞术检测hDPSCs表面抗原CD73、CD90、CD105、CD34表达情况。将hDPSCs分为Ct组、si-NC组、si-GAS5组、pcDNA组、pcDNA-GAS5组、pcDNA-GAS5+mimic NC组、pcDNA-GAS5+miR-103mimic组,qRT-PCR检测hDPSCs中GAS5、miR-103表达;CCK-8法检测hDPSCs增殖能力;取经成牙骨质分化培养基诱导7 d后的hDPSCs,Western blot检测成牙骨质细胞标志蛋白牙骨质附着蛋白(CAP)、牙骨质蛋白1(CEMP1)、骨钙素(OCN)的表达,茜素红S染色检测矿化结节形成;双荧光素酶报告基因实验验证GAS5与miR-103的关系。结果:P3代hDPSCs生长状态佳,细胞呈漩涡状或放射状排列。P3代hDPSCs高表达CD73(99.05%)、CD90(98.27%)、CD105(97.88%),低表达CD34(2.02%),证明成功分离出hDPSCs。与Ct组、si-NC组比较,si-GAS5组hDPSCs中GAS5表达、OD_(450nm)值(3 d、5 d)、CAP、CEMP1、OCN蛋白表达、矿化结节形成率降低,miR-103表达升高(P<0.05);与Ct组、pcDNA组比较,pcDNA-GAS5组hDPSCs中GAS5表达、、OD_(450nm)值(3 d、5 d)、CAP、CEMP1、OCN蛋白表达、矿化结节形成率升高,miR-103表达降低(P<0.05);与pcDNA-GAS5组、pcDNA-GAS5+mimic NC组比较,pcDNA-GAS5+miR-103mimic组hDPSCs中GAS5表达变化差异不显著(P>0.05),OD_(450nm)值(3 d、5 d)、CAP、CEMP1、OCN蛋白表达、矿化结节形成率降低,miR-103表达升高(P<0.05)。结论:过表达GAS5可能通过下调miR-103促进hDPSCs向成牙骨质分化。
Objective:To investigate the influence of long non-coding RNA(lncRNA)growth arrest-specific transcript 5(GAS5)on the differentiation of human dental pulp stem cells(hDPSCs)into cementoblasts by targeting miR-103.Methods:HDPSCs were isolated and cultured from adult third molars,the morphology of P3 generation hDPSCs was observed by inverted optical microscope,the expression of hDPSCs surface antigens CD73,CD90,CD105 and CD34 was detected by flow cytometry.The hDPSCs were separated into Ct group,si-NC group,si-GAS5 group,pcDNA group,pcDNA-GAS5 group,pcDNA-GAS5+mimic NC group,and pcDNA-GAS5+miR-103mimic group.The expression of GAS5 and miR-103 in hDPSCs was detected by qRT-PCR;the proliferation ability of hDPSCs was detected by CCK-8 method;the hDPSCs induced by cementoblast differentiation medium for 7 days were collected.Western blot was used to detect the expression of cementoblast marker proteins cementum attachment protein(CAP),cementum protein 1(CEMP1)and osteocalcin(OCN),alizarin red S staining was used to detect the formation of mineralized nodules.The relationship between GAS5 and miR-103 was verified by dual-luciferase reporter gene experiments.Results:The hDPSCs of the P3 generation grew well,and the cells were arranged in a swirling or radial pattern.P3 generation hDPSCs highly expressed CD73(99.05%),CD90(98.27%),CD105(97.88%),and lowly expressed CD34(2.02%),which proved that hDPSCs were successfully isolated.Compared with Ct group and si-NC group,the GAS5 expression,OD_(450nm)value(3 d,5 d),CAP,CEMP1,OCN protein expression,and mineralized nodule formation rate in hDPSCs of si-GAS5 group were decreased,the expression of miR-103 was increased(P<0.05).Compared with Ct group and pcDNA group,the GAS5 expression,OD_(450nm)value(3 d,5 d),CAP,CEMP1,OCN protein expression,and mineralized nodule formation rate in hDPSCs of pcDNA-GAS5 group were increased,the expression of miR-103 was decreased(P<0.05).Compared with pcDNA-GAS5 group and pcDNA-GAS5+mimic NC group,there was no obvious difference in the expression of GAS5 in hDPSCs of pcDNA-GAS5+miR-103mimic group(P>0.05),the OD_(450nm)value(3 d,5 d),CAP,CEMP1,OCN protein expression,and mineralized nodule formation rate in hDPSCs were decreased,the expression of miR-103 was increased(P<0.05).Conclusion:Overexpression of GAS5 may promote the differentiation of hDPSCs into cementoblasts by down-regulating miR-103.
作者
黄毅斌
郑雅茹
黄丽云
HUANG Yi-bin;ZHENG Ya-ru;HUANG Li-yun(Department of Stomatology of the 909th Hospital of the joint logistics support force(the Southeast Hospital Affiliated to Xiamen University),Fujian Zhangzhou 363000,China)
出处
《临床口腔医学杂志》
2023年第5期273-278,共6页
Journal of Clinical Stomatology
