大鼠成骨细胞膜片的体外构建及成骨潜力评估  

Construction and Osteogenic Potential Evaluation of Rat Osteoblast Cell Sheet in Vitro

作　　者：杨红 夏茜 董强 周建奖[3] YANG Hong;XIA Qian;DONG Qiang;ZHOU Jian-jiang(Department of Prosthodontics,Guiyang Hospital of Stomatology,Guiyang Guizhou,550004;Department of Children's Stomatology,Stomatological Hospital Affiliated to Guizhou Medical University,Guiyang Guizhou,550004;Key Laboratory of Molecular Biology,Guizhou Medical University,Guiyang Guizhou,550004)

机构地区：[1]贵阳市口腔医院修复科,贵州贵阳550004 [2]贵州医科大学附属口腔医院儿童口腔科,贵州贵阳550004 [3]贵州医科大学分子生物学重点实验室,贵州贵阳550004

出　　处：《山西大同大学学报（自然科学版）》2023年第2期52-56,共5页Journal of Shanxi Datong University(Natural Science Edition)

基　　金：国家自然科学基金项目[NS2018-64];贵阳市科技局基金项目[GY2017-43]。

摘　　要：目的体外构建大鼠成骨细胞膜片,并评估其成骨潜力。方法原代培养大鼠成骨细胞鉴定后,高密度连续培养,在维生素C诱导下形成细胞膜片。用扫描电镜观察膜片形貌特征及细胞形态;行碱性磷酸酶(ALP)活性检测;实时荧光定量PCR检测其成骨相关基因I型胶原(COLⅠ)、骨钙素(OC)的表达情况。结果维生素C诱导高密度连续培养成骨细胞5d能获得可机械刮起的成骨细胞膜片。电镜下,由多层细胞组成膜片,细胞层之间有间隙。ALP含量实验组高于对照组,第2周低于第1周。实验组I型胶原mRNA表达情况,第1周与对照组无差异,第2周与第3周高于对照组;实验组内,第1周与第2周无差异,第3周下调。骨钙素mRNA表达,实验组高于对照组;实验组内,表达量随培养时间延长而上调。结论维生素C促进连续培养的成骨细胞形成膜片,机械刮取法能够完整获得。形成的细胞膜片2周内可能生存力较强,可能具备一定成骨潜能。Objective To construct rat osteoblast cell sheets in vitro and evaluate its osteogenic potential.Methods Primary rat osteoblasts were cultured,cell sheets were formed by high-density continuous culture under the induction of vitamin C after identification of osteoblasts.Scanning electron microscopy was used to observe the morphology and cell morphology of the cell sheets.The alkaline phosphatase(ALP)activity in the cell sheets was detected;the expressions of COL I and OC genes,which are osteogenic genes,were detected by real-time fluorescence quantitative PCR.Results Cell sheets were formed by high-density continuous culture for 5 days under the induction of vitamin C,it can be scraped mechanically.Electron microscope observation:the cell sheets is made up of several layers of cells,there are gaps between the the cell layers.The ALP content in the experimental group was higher than that in the control group,and was lower in the second week than in the first week.The expression of type I collagen mRNA in the experimental group was no different from that in the control group at week 1,but higher than that in the control group at week 2 and week 3;In the experimental group,there was no difference between the first week and the second week,but decreased in the third week.The expression of osteocalcin mRNA in the experimental group was higher than that in the control group;In the experimental group,the expression level increased with the prolongation of culture time.Conclusion Vitamin C stimulates the formation of osteoblast cell sheets in continuous culture,the mechanical scraping method can be used to completely obtain them.The osteoblast cell sheets have strong viability and may have osteogenic potential within 2 weeks.

关 键 词：成骨细胞膜片 维生素C ALP COLⅠ OC 

分 类 号：R3[医药卫生—基础医学]