本文選題：M-CSF + 細(xì)胞周期　； 參考：《南華大學(xué)》2007年碩士論文

【摘要】： 目的:建立一個(gè)穩(wěn)定高表達(dá)胞質(zhì)M-CSF的細(xì)胞系,并以此細(xì)胞系為模型,探討胞質(zhì)M-CSF對細(xì)胞增殖、運(yùn)動(dòng)和侵襲能力的影響及其作用機(jī)制。 方法:分別將胞質(zhì)定位空載體pCMV/cyto/myc與重組載體pCMV/cyto/myc- M-CSF穩(wěn)定轉(zhuǎn)染HeLa細(xì)胞,用G418篩選陽性克隆,RT-PCR、Western blot鑒定M-CSF的表達(dá);免疫細(xì)胞化學(xué)驗(yàn)證M-CSF蛋白的定位。倒置顯微鏡下觀察細(xì)胞的形態(tài);細(xì)胞計(jì)數(shù)、MTT法及反義寡核苷酸抑制實(shí)驗(yàn)分析M-CSF對細(xì)胞增殖的影響;半定量RT-PCR觀察胞質(zhì)M-CSF對G1期細(xì)胞周期相關(guān)蛋白、Rho GTP酶(Rac1)基因、基質(zhì)金屬蛋白酶(MMP2、MMP9)的轉(zhuǎn)錄的影響;體外細(xì)胞遷移和侵襲實(shí)驗(yàn)顯示胞質(zhì)M-CSF對HeLa細(xì)胞遷移和侵襲的影響;免疫熒光觀察cdc42和細(xì)胞骨架的改變;明膠酶譜實(shí)驗(yàn)檢測活性MMP的表達(dá)。 結(jié)果: RT-PCR、Western blot實(shí)驗(yàn)顯示:轉(zhuǎn)染M-CSF的HeLa細(xì)胞(命名為HeLa-M細(xì)胞)高表達(dá)M-CSF,與轉(zhuǎn)染空載體HeLa細(xì)胞(命名為HeLa-C細(xì)胞)和HeLa細(xì)胞比較有顯著差異性(P 0.01),免疫細(xì)胞化學(xué)顯示:HeLa-M細(xì)胞表達(dá)的M-CSF蛋白定位于胞質(zhì),提示:實(shí)驗(yàn)成功建立了一個(gè)穩(wěn)定表達(dá)胞質(zhì)M-CSF的HeLa細(xì)胞系。與HeLa-C細(xì)胞和HeLa細(xì)胞比較,HeLa-M細(xì)胞體積增大、倍增時(shí)間縮短、生長速度增快, M-CSF特異性反義寡核苷酸能抑制HeLa-M細(xì)胞的增殖速度,但對HeLa-C細(xì)胞和HeLa細(xì)胞增殖影響較小,提示:HeLa-M細(xì)胞增殖速度增快與細(xì)胞胞質(zhì)M-CSF相關(guān)。HeLa-M細(xì)胞微管蛋白在胞核周呈圓環(huán)狀,放射狀排列,較粗厚,而HeLa-C細(xì)胞和HeLa細(xì)胞微管蛋白彌漫分布呈細(xì)絲狀。HeLa-M細(xì)胞的cyclinE/D1/D3、CDK2/4/6、Rho GTP酶(Rac1)、Rho GTP酶相關(guān)蛋白cdc42和基質(zhì)金屬蛋白酶(MMP2)表達(dá)顯著升高(P 0.01),但cyclinD2、基質(zhì)金屬蛋白酶(MMP9)的表達(dá)沒有明顯變化。Transwell體外細(xì)胞遷移實(shí)驗(yàn)顯示:體外細(xì)胞遷移和侵襲實(shí)驗(yàn)發(fā)現(xiàn)轉(zhuǎn)染組HeLa細(xì)胞遷移和侵襲能力均明顯高于對照組(P 0.01);明膠酶譜實(shí)驗(yàn)發(fā)現(xiàn)轉(zhuǎn)染組細(xì)胞能顯著增強(qiáng)胞外活性MMP2的分泌(P 0.01)。 結(jié)論:建立了一個(gè)穩(wěn)定高表達(dá)胞質(zhì)M-CSF的細(xì)胞系。胞質(zhì)M-CSF上調(diào)cyclinE/D1/D3、CDK2/4/6的表達(dá)、促進(jìn)HeLa細(xì)胞的增殖;上調(diào)Rac1和cdc42的表達(dá)并誘導(dǎo)細(xì)胞微管蛋白的重構(gòu);上調(diào)MMP2的表達(dá)、增強(qiáng)MMP2的活性、誘導(dǎo)HeLa細(xì)胞遷移和侵襲能力的增強(qiáng);胞質(zhì)M-CSF對HeLa細(xì)胞MMP9和cyclinD2表達(dá)的影響較小。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R363

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本文編號：1857706