【作者】 刘磊玉；

【导师】 唐圣松；

【作者基本信息】 南华大学 ， 病理学， 2006， 硕士

【摘要】 目的：建立胞质内稳定表达M-CSF的细胞系；探讨定位在胞质内的M-CSF对细胞增殖和运动的影响。 方法：PCR分别扩增人M-CSF的胞外活性区和功能区，连接到胞浆定位载体pCMV／cyto／myc质粒，得重组质粒pCMV／cyto／myc-h-M-CSF，再将重组质粒稳定转染NIH 3T3细胞，得到分别稳定表达M-CSF的胞外活性区和功能区的细胞株。免疫细胞化学验证M-CSF蛋白的定位，细胞生长曲线和致伤愈合实验显示外源的M-CSF对NIH3T3细胞增殖和运动的影响。考马斯亮兰对细胞骨架进行染色。 结果：重组基因的插入片段大小分别与M-CSF的胞外区和功能区相符，重组的M-CSF片段无阅读框移位和突变，绿色荧光蛋白仅定位在NIH3T3细胞质内，表明成功构建胞浆定位表达载体pCMV／cyto／myc-M-CSF-a和pCMV／cyto／myc-M-CSF-d；分别将pCMV／cyto／myc-M-CSF-a和pCMV／cyto／myc-M-CSF-d质粒转染NIH3T3细胞，并经G418筛选后，稳定表达M-CSF胞外区和功能区的NIH 3T3细胞株其倍增时间均短于对照细胞；胞质内M-CSF诱导NIH3T3细胞微丝重构和细胞迁移；M-CSF N末端149个氨基酸残基组成的功能区其促NIH3T3细胞增殖和运动活性与M-CSF的胞外区基本相同。 结论：建立了一个细胞质稳定表达M-CSF的细胞系；细胞质M-CSF能诱导NIH 3T3细胞增殖，增强细胞运动能力；M-CSF蛋白的功能区诱导细胞增殖与迁移的生物学活性与M-CSF胞外区的活性基本相同。更多还原

【Abstract】 Objective To establish a cytoplasmic M-CSF-stably-expressing cell line, and to explore the effect of cytoplasmic M-CSF on the proliferation and mobility of NIH 3T3 cells.Methods The encoding region and function domain of M-CSF were amplified by PCR respectively and inserted into the cytoplasm localization vector pCMV/cyto/myc to get recombination plasmid pCMV/cyto/myc-h-M-CSF. The cell line, which express stably the encoding region and domain of M-CSF, respectively, was obtained by stable transfection of the recombination plasmid into NIH 3T3 cell. Localization of M-CSF protein was detected by immunocytochemistry. The effect cytoplasmic M-CSF on the proliferation and mobility of NIH 3T3 cells were analyzed by cell conuting and wound healing assay. The cytoskeleton was stained by Coomassie brilliant blue.Results The results show that the size of inserted fragment in the recombinant genes is corresponded to that of both extracellular region and function domain of M-CSF, and that there is no reading frame shift and mutation in recombinant M-CSF and that green fluorescence protein only localizes to the cytoplasm in NIH 3T3 cells, which suggest that M-CSF expression vectors in cytoplasm (pCMV/myc/cyto-M-CSF-a and pCMV/myc/cyto-M-CSF-d) successfully constructed. After transfecting pCMV/myc/cyto-M-CSF-a or pCMV/myc/cyto-M-CSF-d into NIH 3T3 cells, respectively, and screening using G418, our results indicate that both M-CSF-a-and M-CSF-d-expressing NIH 3T3 cells have a shorter doubling time than control cells, and that cytoplasmic M-CSF induce the microfilament reorganization and mobility of NIH 3T3 cells. The activities of function domain of M-CSF, which contains only 149 amino residual in N-terminal in M-CSF, are similar that of the extracellular region of M-CSF in stimulating the proliferation and mobility of NIH 3T3 cells.Conclusion A cell line that express stably cytoplasmic M-CSF was established. Cytoplasmic M-CSF induces the NIH 3T3 cell proliferation and stimulates NIH 3T3 cell更多还原