核转染技术转染大鼠胚胎干细胞的初步研究  

Preliminary Study on Nucleofection of Rat Embryonic Stem Cells

作　　者：吴兆强[1] 赵丽华[1] 张曼玲[1] 金永[1] 聂晓伟[1] 侯道荣[1] 杨宁[1] 陈袁[1] 李荣凤[1] 

机构地区：[1]南京医科大学江苏省异种移植重点实验室,南京210029

出　　处：《中国细胞生物学学报》2015年第3期370-377,共8页Chinese Journal of Cell Biology

基　　金：国家自然科学基金面上项目(批准号:31371487)资助的课题~~

摘　　要：大鼠胚胎干细胞(rat embryonic stem cells,r ESCs)的基因转染研究仍处于探索阶段。该文利用不同的核转染条件,将红色荧光蛋白(red fluorescent protein,RFP)表达载体p EF1alphaDs Red-Express2导入大鼠胚胎干细胞中,比较不同转染条件下大鼠胚胎干细胞的红荧光蛋白表达效率和死亡率,确定最佳核转染条件。在采用最佳核转染条件转染大鼠胚胎干细胞后,利用碱性磷酸酶染色、RT-PCR、免疫荧光染色等方法,比较核转染前后大鼠胚胎干细胞的干细胞特性,探讨核转染过程对大鼠胚胎干细胞的干细胞特性的影响。实验结果显示,转染条件为A-13时,可获得最好的大鼠胚胎干细胞转染效率(39.63±1.75)%。核转染后,表达红色荧光蛋白的大鼠胚胎干细胞的碱性磷酸酶染色结果仍为阳性。另外,RT-PCR和免疫荧光染色结果表明,核转染前后大鼠胚胎干细胞的干细胞多能性标志基因的表达情况未出现明显差异。研究表明,核转染条件A-13可以在不改变大鼠胚胎干细胞特性的条件下有效地对其进行基因转染,即核转染技术适用于大鼠胚胎干细胞的基因转染。The study of gene transduction on rat embryonic stem cells（r ESCs） has been explored continually. In our study, in order to detect the optimized program for gene transduction, the reporter red fluorescent protein（RFP） gene was transfected into r ESCs with different nucleofection programs. The proportion of cells with red fluorescent and the dead cells after transfection were compared among different programs. Then, the best program was selected and used to transfect r ESCs. Alkaline phosphatase（AP） staining, RT-PCR and immunostaining were performed to compare the characters of r ESCs before and after nucleofection. The results showed that the A-13 was the best program for r ESCs transfection and its efficiency reached（39.63±1.75）%. The AP staining of r ESCs was still positive after nucleofection, and the expression and immunostaining of ESC marker genes（RT-PCR and immunostaining） were not changed by the nucleofection process. Therefore, the r ESCs could be effectively transducted with the nucleofection program A-13 and their morphology and pluripotency characters were maintained. Nucleofection was an effective method for gene transduction of r ESCs.

关 键 词：大鼠胚胎干细胞 红色荧光蛋白 核转染 干细胞多能性标志基因 

分 类 号：Q78[生物学—分子生物学]