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Objective To explore the effects and mechanisms of cytoplasmic M-CSF on the proliferation,migration and invasion of HeLa cells.Methods Both pCMV/cyto/myc vector and pCMV/cyto/myc-M-CSF vector was transfected into HeLa-cell by transfectaimine.After screening by G418,the positive clones were amplified and confirmed by RT-PCR,Western blot and immunocytochemistry.The effect of cytoplasmic M-CSF on the proliferation of HeLa cells were analyzed by cell conuting and antisense oligonucleotides.The migration and invasion of cell was measured by in vitro Transwell assay and Matrigel-coated polycarbonate filters.The expression of cyclinE,cyclinD1/2/3,CDK2/4/6,Rac1,and matrix metalloproteinase 2 and 9(MMP2/9)were assayed by semiquantitative RT-PCR.And expression of both α-tubulin and cdc42 were displayed by immunofluorescence.The activity of MMP2 was detected by gelatin zymography.Results Results A cell line(referred as to HeLa-M cell)that highly expresses cytoplasmic M-CSF was successfully established in the test.Our result indicated that HeLa-M cell had a larger volume,faster growth rate and shorter doubling time than either pCMV/cyto/myc transfected HeLa cells(referred as to HeLa-C cell)or untransfected HeLa cells(referred as to HeLa cell).M-CSF-specific antisense oligonucleoside significantly inhibited HeLa-M cell proliferation and had little effect on either HeLa-C cell or HeLa-C cell growth.Cytoplasmic M-CSF up-regulated both the expression of cyclinE,cyclinD1 and cyclinD3,CDK2,CDK 4 and CDK6,a Rho GTPase ralative protein(Rac1),cdc42 and MMP2,but had little effect on expression of MMP9 and cyclin D2.Furthermore,cytoplasmic M-CSF induced the rearrangement of the α-tubulin in HeLa cells and significantly promoted the migration and invasion of HeLa cells in vitro.Conclusions Cytoplasmic M-CSFs up-regulate the expression of cyclinE,cyclinD1 and cyclinD3,CDK2,CDK 4 and CDK6 and induces the proliferation of HeLa cells.Cytoplasmic M-CSFs up-regulate the expression of Rac1 and cdc42 and cause the rearrangement of the α-tubulin in HeLa cells.Furthermore Cytoplasmic M-CSFs increase both the expression and activity of MMP2 and promote the migration and invasion of HeLa cell in vitro.But cytoplasmic M-CSFs have little effect on expression of cyclin D2 and MMP9.
Objective To explore the effects and mechanisms of cytoplasmic M-CSF on the proliferation, migration and invasion of HeLa cells. Methods Both pCMV / cyto / myc vector and pCMV / cyto / myc-M-CSF vector was transfected into HeLa-cell by transfectaimine . After screening by G418, the positive clones were amplified and confirmed by RT-PCR, Western blot and immunocytochemistry. The effect of cytoplasmic M-CSF on the proliferation of HeLa cells were analyzed by cell conuting and antisense oligonucleotides. The migration and invasion of cell was measured by in vitro Transwell assay and Matrigel-coated polycarbonate filters. The expression of cyclinE, cyclinD1 / 2/3, CDK2 / 4/6, Rac1, and matrix metalloproteinase 2 and 9 (MMP2 / 9) were assayed by semiquantitative RT -PCR.And expression of both α-tubulin and cdc42 were displayed by immunofluorescence. The activity of MMP2 was detected by gelatin zymography. Results Results A cell line (referred to as HeLa-M cell) that highly explassed cytoplasmic M-CSF was successfully estab lished in the test. Our result indicates that HeLa-M cell had a larger volume, faster growth rate and shorter doubling time than either pCMV / cyto / myc transfected HeLa cells (referred to to HeLa-C cell) or untransfected HeLa cells (referred to as to HeLa cell) .M-CSF-specific antisense oligonucleoside significantly inhibited HeLa-M cell proliferation and had little effect on either HeLa-C or HeLa-C cell growth. Cytoplasmic M-CSF up-regulated both the expression of cyclinE, cyclinD1 and cyclinD3, CDK2, CDK4 and CDK6, a Rho GTPase ralative protein (Rac1), cdc42 and MMP2, but had little effect on the expression of MMP9 and cyclin D2.Furthermore, cytoplasmic M-CSF induced the rearrangement of the α-tubulin in HeLa cells and enhanced promoted the migration and invasion of HeLa cells in vitro. Conclusions Cytoplasmic M-CSFs up-regulate the expression of cyclin E, cyclinD1 and cyclinD3, CDK2, CDK4 and CDK6 and induces the proliferation of HeLa cells. Cytoplasmic M- CSFs up-regulate the expression of Rac1 and cdc42 and cause the rearrangement of the α-tubulin in HeLa cells. Further Thermo-Cytoplasmic M-CSFs increase both the expression and activity of MMP2 and promote the migration and invasion of HeLa cells in vitro. Bute cytoplasmic M-CSFs have little effect on expression of cyclin D2 and MMP9.