| 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... |
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