【作者】 赵雪琴；

【导师】 唐圣松；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2006， 硕士

【摘要】 目的：建立M-CSF核内稳定表达细胞系，探讨核内M-CSF对Cos7细胞增殖的影响，鉴定M-CSF核内相互作用靶分子。 方法：用PCR技术扩增M-CSF活性区，并插入真核表达质粒pCMV／myc／nuc，构建M-CSF核内定位重组表达质粒pCMV／nuc／M-CSF。经琼脂糖凝胶电泳、PCR、限制性酶切和测序鉴定重组子后，用脂质体分别将pCMV／nuc／M-CSF、pCMV／myc／nuc和pCMV／nuc／GFP转染Cos7细胞，经G418筛选并扩增阳性克隆后，用RT-PCR、免疫细胞化学和Western blot证实阳性克隆细胞是否稳定表达M-CSF。用细胞计数和MTT法观察核内M-CSF对细胞增殖的影响，用免疫共沉淀鉴定M-CSF的核内相互作用分子。 结果：琼脂糖凝胶电泳与限制性酶切分析显示插入质粒的片段大小与M-CSF分子大小相当，DAN测序分析表明插入质粒的M-CSF无读码框移位和突变。荧光倒置显微镜观察绿色荧光只定位于细胞核内，RT-PCR分析显示pCMV／nuc／M-CSF转染细胞表达M-CSF mRNA，免疫细胞化学与Western blot显示M-CSF准确定位于pCMV／nuc／M-CSF转染细胞核内。细胞计数显示pCMV／nuc／M-CSF转染细胞、未转染的Cos7细胞和pCMV／myc／nuc转染细胞的倍增时间分别为20.73±0.22h、28.22±0.25h和27.88±0.24h。细胞计数与MTT显示pCMV／nuc／M-CSF转染细胞的生长速率比未转染的Cos7细胞和pCMV／myc／nuc转染细胞更快。用免疫共沉淀分析表明MCM7能被抗M-CSF抗体沉淀。更多还原

【Abstract】 Objective1. To establish a cell line which stably express M-CSF in nucleus.2. To explore the effect of nuclear M-CSF on the proliferation of Cos7 cell, a African green monkey kidney cell line.3. To identify M-CSF-interacting proteins in Cos7 cells.Methods An active M-CSF fragment was amplified by Polymerase chain reaction(PCR) and subcloned to a eukaryotic expression vector (pCMV/myc/nuc) to construct a M-CSF-expressing in nucleus plasmid (pCMV/nuc/M-CSF) by gene recombination. The recombinant gene was identified by agarose gel electrophoresis, restriction enzyme analysis and sequencing. pCMV/nuc/myc, pCMV/nuc/GFP and pCMV/nuc/M-CSF were stably transfected into Cos7 cells using liposome. After screening with G418, the expression and localization of M-CSF in Cos7 cells were verified by fluorescence microscopy, RT-PCR, immunocytochemistry and Western blot. The effect of nuclear M-CSF on Cos7 cell proliferation was analyzed by cell count and MTT method. The M-CSF-interacting proteins were identified by poll-down test.Results The results showed that the size of inserted fragment in the recombinant gene corresponded to that of M-CSF, and that there was no reading frame shifts and mutations in recombinant M-CSF, and that green fluorescence protein only localized to the nucleus in Cos7 cells, which suggested that M-CSF expression vector in nucleus (pCMV/nuc/M-CSF) was successfully constructed. After transfecting pCMV/nuc/M-CSF into Cos7 cells by liposome and screening with G418, Our results indicated that the M-CSF-transfected Cos7 cells stably expressed both M-CSF mRNA and protein in nucleus, which suggested that a更多还原