绿色荧光蛋白基因转染骨髓基质干细胞的瞬时表达检测

Test of Transient Expression of Green Fluorescent Protein Gene Transferred to Bone Marrow Stromal Cells

目的 检测绿色荧光蛋白基因 (EGFP)的表达载体pEGFP对MSCs的转染效率及瞬时表达 ,为利用pEGFP构建骨形成蛋白 (BMP)表达载体并转染骨髓基质干细胞 (MSCs)提供依据。方法 在体外扩增并酶切鉴定pEGFP质粒 ;抽取兔骨髓 ,应用贴壁法培养MSCs;质粒与脂质体按照不同的比例混合 ,以脂质体介导法转染pEGFP ,应用荧光显微镜观察转染效率及瞬时表达情况。结果 转染效率与脂质体和质粒的比例有关 ,绿色荧光蛋白在基因转染 2 4h后开始表达 ,4 8～ 72h最高 ,1周后表达逐渐减弱 ,但 3～ 4周后仍有少量表达。结论 按照合适的质粒和脂质体比例 ,pEGFP转染MSCs的效率可达到 30 % ,并能持续表达 3周以上 ,是转染MSCs较为理想的瞬时表达载体。

Objective\ To determine the optimized condition under which BMP expression vector will be constructed to transfect bone marrow stromal cells (MSCs), plasmid vector coding enhanced green fluorescence protein (EGFP) gene pEGFP was transferred into MSCs. The transfer efficiency and transient expression were subsequently tested. Methods\ pEGFP plasmid was amplified and tested by an enzyme cutting technique in vitro. MSCs, which were initially obtained from the bone marrow of rabbits, were cultured in vitro and transferred with pEGFP by means of lipofectamine media methods. The ratio of plasmid and lipofectamine was varied according to the experiment design. Transfer efficiency and transient expression were evaluated by fluorescent microscopy. Results\ Transfer efficiency was correlated with the ratio of plamid and lipofectamine. The expression of EGFP began in 24 hours after transferring, reached maximum in 48_72 hours and decreased in 1 week, however there remained a weak expression for more than 3 weeks. Conclusion\ The efficiency of transferring pEGFP into MSCs could achieve to 30% with proper ratio of plamid and lipofactamine. pEGFP was an ideal transient expression vector for MSCs gene transference.\;