CAJ | 학술논문

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。
배경：대아수간세포진행은정고효안전적체외표기시시종기술중수선수요해결적문제，야시아치재생체내연구적기출。 　　목적：탐토만병독재체개도록색형광단백전염대서아수간세포적이상조건급방법，병학정기전염후시부보지간세포특성。 　　방법：통과개량매소화법획득대서아수간세포，대기면역표형급분화잠능진행감정，이감염복수위5，10，25，50화100적만병독재체개도록색형광단백작용24 h화48 h，도치현미경하검측전염솔화형광강도，병대대서아수간세포감염전후적극륭증식능력、세포주기급아향분화능력진행비교，평개감염대기생물학특성적영향。 　　결과여결론：류식세포의검측결과현시대서아수간세포STRO-1화CD146표체양성，CD34화CD45표체음성，경상응유도배양후가향성골화성지분화。당감염복수위50，작용시간위48 h시，전염효솔최고，형광표체최강；감염전후세포증식、극륭형성솔급세포주기등방면차이무현저성의의(P>0.05)，감성린산매양성표체。설명감염복수위50작용48 h시만병독재체개도록색형광단백전염대서아수간세포적이상조건，차불영향아수간세포생물학특성，위대서아수간세포적체내연구제공료가고적시종방법。
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. 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 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.