胞质M-CSF对HeLa细胞增殖、运动和侵袭的影响及机制

发布时间：2018-05-07 16:53

  本文选题：M-CSF + 细胞周期　； 参考：《南华大学》2007年硕士论文

【摘要】： 目的:建立一个稳定高表达胞质M-CSF的细胞系,并以此细胞系为模型,探讨胞质M-CSF对细胞增殖、运动和侵袭能力的影响及其作用机制。 方法:分别将胞质定位空载体pCMV/cyto/myc与重组载体pCMV/cyto/myc- M-CSF稳定转染HeLa细胞,用G418筛选阳性克隆,RT-PCR、Western blot鉴定M-CSF的表达;免疫细胞化学验证M-CSF蛋白的定位。倒置显微镜下观察细胞的形态;细胞计数、MTT法及反义寡核苷酸抑制实验分析M-CSF对细胞增殖的影响;半定量RT-PCR观察胞质M-CSF对G1期细胞周期相关蛋白、Rho GTP酶(Rac1)基因、基质金属蛋白酶(MMP2、MMP9)的转录的影响;体外细胞迁移和侵袭实验显示胞质M-CSF对HeLa细胞迁移和侵袭的影响;免疫荧光观察cdc42和细胞骨架的改变;明胶酶谱实验检测活性MMP的表达。 结果: RT-PCR、Western blot实验显示:转染M-CSF的HeLa细胞(命名为HeLa-M细胞)高表达M-CSF,与转染空载体HeLa细胞(命名为HeLa-C细胞)和HeLa细胞比较有显著差异性(P 0.01),免疫细胞化学显示:HeLa-M细胞表达的M-CSF蛋白定位于胞质,提示:实验成功建立了一个稳定表达胞质M-CSF的HeLa细胞系。与HeLa-C细胞和HeLa细胞比较,HeLa-M细胞体积增大、倍增时间缩短、生长速度增快, M-CSF特异性反义寡核苷酸能抑制HeLa-M细胞的增殖速度,但对HeLa-C细胞和HeLa细胞增殖影响较小,提示:HeLa-M细胞增殖速度增快与细胞胞质M-CSF相关。HeLa-M细胞微管蛋白在胞核周呈圆环状,放射状排列,较粗厚,而HeLa-C细胞和HeLa细胞微管蛋白弥漫分布呈细丝状。HeLa-M细胞的cyclinE/D1/D3、CDK2/4/6、Rho GTP酶(Rac1)、Rho GTP酶相关蛋白cdc42和基质金属蛋白酶(MMP2)表达显著升高(P 0.01),但cyclinD2、基质金属蛋白酶(MMP9)的表达没有明显变化。Transwell体外细胞迁移实验显示:体外细胞迁移和侵袭实验发现转染组HeLa细胞迁移和侵袭能力均明显高于对照组(P 0.01);明胶酶谱实验发现转染组细胞能显著增强胞外活性MMP2的分泌(P 0.01)。 结论:建立了一个稳定高表达胞质M-CSF的细胞系。胞质M-CSF上调cyclinE/D1/D3、CDK2/4/6的表达、促进HeLa细胞的增殖;上调Rac1和cdc42的表达并诱导细胞微管蛋白的重构;上调MMP2的表达、增强MMP2的活性、诱导HeLa细胞迁移和侵袭能力的增强;胞质M-CSF对HeLa细胞MMP9和cyclinD2表达的影响较小。
[Abstract]:Aim: to establish a cell line with stable and high expression of cytoplasmic M-CSF, and to investigate the effects of cytoplasmic M-CSF on cell proliferation, motion and invasion and its mechanism. Methods: the empty cytoplasmic vector pCMV/cyto/myc and the recombinant vector pCMV / P cytomyc- M-CSF were stably transfected into HeLa cells respectively. The expression of M-CSF was identified by RT-PCR Western blot with G418 positive clone, and the localization of M-CSF protein was confirmed by immunocytochemistry. Cell morphology was observed under inverted microscope, cell count and antisense oligonucleotide inhibition assay were used to analyze the effect of M-CSF on cell proliferation. Semi-quantitative RT-PCR was used to observe the effect of cytoplasmic M-CSF on Rho GTP gene. The effects of matrix metalloproteinase (MMP2) and MMP9) on the expression of cdc42 and cytoskeleton in HeLa cells were observed by in vitro cell migration and invasion assay. The expression of active MMP was detected by gelatinase assay. Results: the expression of M-CSF in M-CSF transfected HeLa cells (named HeLa-M cells) was significantly higher than that in empty vector HeLa cells (named HeLa-C cells) and HeLa cells. The expressed M-CSF protein was located in the cytoplasm. The results suggest that a stable HeLa cell line expressing cytoplasmic M-CSF was successfully established. Compared with HeLa-C cells and HeLa cells, the volume of HeLa-M cells increased, the doubling time shortened and the growth rate increased. M-CSF specific antisense oligonucleotides could inhibit the proliferation of HeLa-M cells, but had little effect on the proliferation of HeLa-C cells and HeLa cells. The results suggest that the increase of cell proliferation speed is related to cytoplasmic M-CSF. The microtubulin of HeLa-M cells is circular, radial and thicker around the nucleus. However, the expression of Rho GTP related protein cdc42 and matrix metalloproteinase (MMP2) in HeLa-C cells and HeLa cells distributed in filamentous. HeLa-M cells was significantly increased, but the expression of cyclin D _ 2 and matrix metalloproteinase MMP9 did not change significantly. Transwell cell migration assay in vitro showed that the migration and invasion ability of HeLa cells in transfection group was significantly higher than that in control group (P 0.01), and gelatinase assay showed that the transfected group could significantly enhance the secretion of extracellular active MMP2 (P0.01). Conclusion: a cell line with stable and high expression of cytoplasmic M-CSF was established. Cytoplasmic M-CSF up-regulated the expression of cyclin E / D _ 1 / D _ 3 / CDK _ 2 / 4 / 6, promoted the proliferation of HeLa cells, up-regulated the expression of Rac1 and cdc42 and induced the remodeling of tubulin, up-regulated the expression of MMP2, enhanced the activity of MMP2, and enhanced the migration and invasion of HeLa cells. Cytoplasmic M-CSF had little effect on the expression of MMP9 and cyclinD2 in HeLa cells.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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