腺病毒载体介导的LacZ基因在神经前体细胞的转染和表达

为了观察含报告基因 L ac Z的重组 5型腺病毒载体 ( Ad5CMVLac Z)在神经前体细胞转染和表达的量效关系并探讨用该载体构建基因修饰细胞的可能性 ,本研究用不同滴度 ( 1× 10 3～ 1× 10 1 0 PFU/ml)的 Ad5CMVLac Z转染体外培养的 SD胎鼠(胎龄 12 d)海马神经前体细胞 ,用β-半乳糖苷酶 (β-gal)免疫组化反应检测转染效率。结果显示 :当病毒滴度为 1× 10 7时 ,转染率约为 5 0 % ,当滴度增加到 1× 10 1 0时 ,转染率达 10 0 %。Ad5CMVL ac Z在神经前体细胞的转染率具有滴度依赖性的量效关系。表明高滴度的 Ad5CMVLac Z成功地转染了大部分神经前体细胞 ,Lac Z基因也得到充分表达。提示神经前体细胞是该载体转染的适宜靶细胞 ,并有可能通过该载体转导目的基因 ,构建出基因修饰细胞

ADENOVIRUS-MEDIATED LacZ GENE TRANSFECTION AND EXPRESSION IN CULTURED NEURAL PRECURSOR CELLS

In order to observe the dose effect relationship of transfection and expression of recombinant adenoviral vector (Ad 5 CMVLacZ) in neural precursor cells (NPCs) and to explore the possibility of constructing genetically engineered NPCs with the vector, in vitro cultured NPCs derived from hippocampus in SD rat embryos (E12) were transfected by various titers (1×10 3～1×10 10 PFU/ml) of Ad 5 CMVLacZ. The expression of Ad 5 CMVLacZ in the transfected NPCs were detected by β galactosidase (β gal) immunohistochemical analyses. In cultured NPCs, the transfective rate was about 50% when the titer of Ad 5 CMVLacZ was 1×10 7 PFU/ml;the rate reached 100% when the titer increased to 1×10 10 PFU/ml. The transfective efficiency of Ad 5 CMVLacZ in cultured NPCs showed the dose effect relationship with titer dependence. The above results suggest that the majority of cultured NPCs are transfected by Ad 5 CMVLacZ with higher titers (1×10 7～1×10 10 PFU/ml), and LacZ gene in the transfected NPCs can fully be expressed, indicating that NPC may be a proper candidate for preparing genetically engineered cells with the recombinant adenoviral vector.