摘要
背景:研究表明,小鼠前破骨细胞分泌的血小板衍生生长因子BB可以促进成骨细胞和成血管细胞形成,尚无血小板衍生生长因子BB是否可以促进骨髓间充质干细胞向成骨细胞分化的相关研究。目的:探讨血小板衍生生长因子BB诱导大鼠骨髓间充质干细胞向成骨细胞分化的可行性。方法:①体外分离培养SD大鼠骨髓间充质干细胞,第3代细胞经流式鉴定其表面标记物CD45、CD29和CD34的表达,选用纯度较高的骨髓间充质干细胞进行血小板衍生生长因子BB干预实验;②第3代骨髓间充质干细胞经25μg/L血小板衍生生长因子BB诱导3 d,进行Ⅰ型胶原免疫荧光染色,然后采用碱性磷酸酶改良钙钴法和偶氮偶联法、茜素红法对细胞进行染色,鉴定成骨细胞的表面特征,最后采用RT-PCR检测Runx-2和Gli-2的mRNA水平。结果与结论:①经细胞免疫荧光染色,诱导后细胞的Ⅰ型胶原绿色荧光表达率>90%;②经碱性磷酸酶改良钙钴法染色,可见细胞胞质中有黑色条索状附着,说明这些细胞均表达有活性的碱性磷酸酶;经碱性磷酸酶偶氮偶联法染色,可见细胞胞浆中含有蓝色的有活性的碱性磷酸酶;经茜素红染色,可见细胞聚集在一起形成小的红色的钙结节;③经RT-PCR检测,血小板衍生生长因子BB诱导后Gli-2和Runx-2的mRNA表达均高于未诱导骨髓间充质干细胞(P<0.05);④结果表明,大鼠骨髓间充质干细胞经血小板衍生生长因子BB诱导后可以分化为具有功能的成骨细胞。
BACKGROUND:It has been shown that platelet-derived growth factor-BB secreted by mouse osteoclasts can promote the formation of osteoblasts and vascular cells.Whether platelet-derived growth factor-BB can promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts has not been studied.OBJECTIVE:To investigate the feasibility of platelet-derived growth factor-BB on inducing bone marrow mesenchymal stem cells of rats into osteoblasts.METHODS:(1)The bone marrow mesenchymal stem cells of Sprague-Dawley rats were isolated and cultured in vitro.The surface markers CD45,CD29 and CD34 of the third generation were identified by flow cytometry.Bone marrow mesenchymal stem cells with high purity were selected for platelet-derived growth factor-BB intervention experiments.(2)Bone marrow mesenchymal stem cells at passage 3 were induced by 25μg/L platelet-derived growth factor-BB for 3 days.Cellular immunofluorescence was used to identify type I collagen,followed by alkaline phosphatase modified calcium-cobalt method and azo coupling method kit,alizarin red method.The surface characteristics of osteoblasts were identified.Finally,RT-PCR was used to detect the mRNA levels of Runx-2 and Gli-2 genes.RESULTS AND CONCLUSION:(1)Cellular immunofluorescence staining showed that the green fluorescence expression rate of type I collagen was more than 90%after induction.(2)Alkaline phosphatase modified calcium-cobalt method demonstrated that black cords were attached to the cytoplasm of the cells,indicating that these cells expressed active alkaline phosphatase.Alkaline phosphatase azo coupling method showed that blue active alkaline phosphatase was found in the cytoplasm of cells.Alizarin red staining exhibited that induced cells gathered together to form small red calcium nodules.(3)RT-PCR showed that the mRNA levels of Gli-2 and Runx-2 genes were higher than those in bone marrow mesenchymal stem cells after platelet-derived growth factor-BB induction(P<0.05).(4)It is concluded that rat bone marrow mesenchymal st
作者
魏琴
张雪
马磊
李志强
寿玺
段明军
吴硕
贾麒钰
马创
Wei Qin;Zhang Xue;Ma Lei;Li Zhiqiang;Shou Xi;Duan Mingjun;Wu Shuo;Jia Qiyu;Ma Chuang(Laboratory Animal Center of Xinjiang Medical University,Xinjiang Key Laboratory of Medical Animal Model Research,Urumqi 830011,Xinjiang Uygur Autonomous Region,China;Clinical Medical Research Institute of First Affiliated Hospital,Xinjiang Medical University,Urumqi 830054,Xinjiang Uygur Autonomous Region,China;Department of Orthopedics,Second Affiliated Hospital of Xinjiang Medical University,Urumqi 830000,Xinjiang Uygur Autonomous Region,China;Department of Microrestorative Surgery,Department of Orthopedics Center,First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,Xinjiang Uygur Autonomous Region,China)
出处
《中国组织工程研究》
CAS
北大核心
2021年第19期2953-2957,共5页
Chinese Journal of Tissue Engineering Research
基金
新疆维吾尔自治区高校科研计划(XJEDU2020Y022),项目负责人:魏琴
国家自然科学基金(81760397),项目负责人:马创。
关键词
干细胞
骨髓间充质干细胞
血小板衍生生长因子BB
成骨细胞
钙结节
碱性磷酸酶
stem cells
bone marrow mesenchymal stem cells
platelet-derived growth factor BB
osteoblasts
calcium nodules
alkaline phosphatase
