核内M-CSF对Cos7细胞增殖的影响及其靶分子的鉴定

发布时间：2018-01-11 12:00

本文关键词：核内M-CSF对Cos7细胞增殖的影响及其靶分子的鉴定　出处：《南华大学》2006年硕士论文　论文类型：学位论文

【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。
[Abstract]:Objective: to establish a stable expression cell line in M-CSF nucleus, to explore the effect of M-CSF on the proliferation of Cos7 cells, and to identify the interaction target molecules in the M-CSF nucleus.
Methods: the amplification of M-CSF activity by PCR, and inserted into the eukaryotic expression plasmid pCMV / myc / NUC, construction of M-CSF nuclear localization of recombinant expression plasmid pCMV / NUC / M-CSF. by agarose gel electrophoresis, PCR, restriction enzyme digestion and sequencing after recombinant plasmid by liposome, respectively pCMV / NUC / M-CSF. PCMV / myc / NUC and pCMV / NUC / GFP was transfected into Cos7 cells, RT-PCR were screened by G418 and amplified after positive clones, immunocytochemistry and Western blot confirmed positive clone cells with stable expression of M-CSF. by cell counting and MTT method to observe the effect of nuclear M-CSF on cell proliferation, nuclear interactions, molecular identification of M-CSF by co immunoprecipitation.
Results: agarose gel electrophoresis and restriction analysis showed that a fragment size and the molecular size of the M-CSF is inserted into the plasmid DAN and inserted into the plasmid M-CSF sequencing analysis showed that the open reading frame shift mutation. And the fluorescence microscope observation of green fluorescence only localized in the nucleus, RT-PCR analysis showed that pCMV / NUC / M-CSF transfected cells expressing M-CSF mRNA Western, immunocytochemistry and blot showed that M-CSF is accurately located in the pCMV / NUC / M-CSF was transfected into cell nucleus. Cells show that pCMV / NUC / M-CSF transfected cells, the doubling time of untransfected Cos7 cells and pCMV / myc / NUC transfected cells were 20.73 + 0.22h, 28.22 + 0.25h and 27.88 + 0.24h. cell count and MTT showed that the growth rate of pCMV / NUC / M-CSF transfected cells faster than non transfected Cos7 cells and pCMV / myc / NUC transfected cells. The results show that MCM7 can be used in CO immunoprecipitation Anti M-CSF antibody precipitation.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

【参考文献】