【摘要】 目的构建M-CSF细胞核内定位表达载体pCMV/M-CSF,探讨核内M-CSF对NIH3T3细胞运动的影响。方法采用PCR的方法扩增人M-CSF活性片段,将其插入核内真核表达载体pCMV/myc/nuc,构建重组体pCMV/M-CSF,经脂质体介导转染NIH3T3细胞,G418筛选后,用免疫细胞化学及Western blot鉴定其在真核细胞中的表达及定位分布,用细胞划痕实验测定M-CSF进入细胞核后对细胞运动能力的影响。结果限制性双酶切及DNA测序分析结果显示插入pCMV/M-CSF的片段为1400bp左右,与预期M-CSF分子大小相当。Western blot结果显示转染pCMV/M-CSF的NIH3T3细胞能稳定表达M-CSF蛋白;免疫细胞化学结果显示表达的M-CSF定位于NIH3T3细胞核。细胞划痕实验显示转染pCMV/M-CSF的NIH3T3细胞有较强的运动能力。结论成功构建M-CSF核内定位表达载体pCMV/M-CSF,核内M-CSF可加强NIH3T3细胞运动。更多还原

【Abstract】 Objectives To construct macrophage colony-stimulating factor(M-CSF)-expressing vector (pCMV/M-CSF) and explore the effect of nuclear M-CSF on the NIH3T3 cells movement. Methods Function part of M-CSF cDNA was amplified by PCR and inserted to nucleus-localization expression vector pCMV/myc/nuc to construct pCMV/M-CSF. Then the pCMV/M-CSF was transfected into NIH3T3 cells and the cells clones were selected with G418. The expression and localization of M-CSF in NIH3T3 cells were verified by immunocytochemistry and Western blot. At last,the effect of nuclear M-CSF on cell movement was analyzed by cell scratch assay. Results The size of inserted fragments in the recombinant vectors pCMV/M-CSF is 1400bp, corresponding to that of M-CSF. After transfecting pCMV/M-CSF into NIN3T3 cells by liposome and screening using G418, pCMV/M-CSF-transfected NIN3T3 cells can stably express M-CSF protein in nucleus. The results from cell scratch assay show that pCMV/M-CSF-transfected NIN3T3 cells have stronger movement ability. Conclusions The nuclear localization vector pCMV/M-CSF was successfully constructed, M-CSF in nucleus promoted the NIH3T3 cells movement.更多还原