慢病毒载体介导绿色荧光蛋白转染大鼠牙髓干细胞  

Transfection of stem cells derived from rat dental pulp with green fluorescent protein infection by lentiviral vector

作　　者：张瑞涵[1] 刘佳[1] 聂姗姗[1] 王璇[1] 李伯琦[1] 孙大磊[1] 热甫卡提.地力毛拉提 刘奕杉[1] 

机构地区：[1]新疆医科大学第一附属医院儿童口腔科-口腔预防科,新疆维吾尔自治区乌鲁木齐市830054

出　　处：《中国组织工程研究》2014年第45期7299-7305,共7页Chinese Journal of Tissue Engineering Research

基　　金：新疆维吾尔自治区自然科学基金面上项目(2011211A067)~~

摘　　要：背景:对牙髓干细胞进行稳定高效安全的体外标记是示踪技术中首先需要解决的问题,也是牙齿再生体内研究的基础。目的:探讨慢病毒载体介导绿色荧光蛋白转染大鼠牙髓干细胞的理想条件及方法,并确定其转染后是否保持干细胞特性。方法:通过改良酶消化法获得大鼠牙髓干细胞,对其免疫表型及分化潜能进行鉴定,以感染复数为5,10,25,50和100的慢病毒载体介导绿色荧光蛋白作用24 h和48 h,倒置显微镜下检测转染率和荧光强度,并对大鼠牙髓干细胞感染前后的克隆增殖能力、细胞周期及牙向分化能力进行比较,评价感染对其生物学特性的影响。结果与结论:流式细胞仪检测结果显示大鼠牙髓干细胞STRO-1和CD146表达阳性,CD34和CD45表达阴性,经相应诱导培养后可向成骨和成脂分化。当感染复数为50,作用时间为48 h时,转染效率最高,荧光表达最强;感染前后细胞增殖、克隆形成率及细胞周期等方面差异无显著性意义(P>0.05),碱性磷酸酶阳性表达。说明感染复数为50作用48 h是慢病毒载体介导绿色荧光蛋白转染大鼠牙髓干细胞的理想条件,且不影响牙髓干细胞生物学特性,为大鼠牙髓干细胞的体内研究提供了可靠的示踪方法。BACKGROUND：Stable and efficient labeling of dental pulp stem cel s in vitro is most important in tracer technique, which is also the basis of tooth regeneration in vivo. OBJECTIVE：To determine the optimal condition and method for transfection of stem cel s derived from rat dental pulp with green fluorescent protein infection by lentiviral vector and to determine whether green fluorescent protein-labeled dental pulp stem cel s maintain their stem cel properties. 〈br〉 METHODS：Rat dental pulp stem cel s were obtained by modified enzyme digestion method, to identify the immune phenotype and differentiation potential. Dental pulp stem cel s were infected with green fluorescent protein by lentiviral vector for 24 and 48 hours at different multiplicity of infection （MOI） （5, 10, 25, 50 and 100）. The infection efficiency and fluorescence intensity were analyzed by inverted fluorescent microscopy. The clonal and proliferation ability, cel cycle and the mineralization potential were compared before and after transfection. Based on those mentioned above, we could evaluate the influence of infection on their biological characteristics. RESULTS AND CONCLUSION：Flow cytometry results showed that rat dental pulp stem cel s expressed STRO-1 and CD146 but not CD34 or CD45. The dental pulp stem cel s could differentiate into osteoblasts and 〈br〉 adipocytes when cultured in specific medium for each lineage differentiation. The highest efficiency of infection and strongest fluorescence expression appeared at 48 hours of infection and MOI 50. There were no significant differences in growth ability, cel colony formation rate and cel cycle before and after transfection （P〉0.05）. And the alkaline phosphatase expressed positively. Infection for 48 hours at MOI 50 is optimal for transfecting dental pulp stem cel s with green fluorescent protein by a lentiviral vector, thereby providing reliable tracer method for the study of rat dental pulp stem cel s in vivo.

关 键 词：干细胞 分化 牙髓干细胞 慢病毒载体 绿色荧光蛋白 大鼠 转染 组织工程 新疆维吾尔自治区自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]