M-CSF在NIH3T3细胞核内的表达及其对细胞运动的影响

发布时间：2018-04-01 03:00

本文选题：巨噬细胞集落刺激因子(M-CSF)　切入点：核内定位载体pCMV/myc/nuc　出处：《南华大学》2006年硕士论文

【摘要】：目的：构建GFP-M-CSF融和蛋白真核表达载体pEGFP／M-CSF，分析M-CSF在NIH3T3细胞内的定位分布；构建M-CSF细胞核内定位表达载体pCMV／M-CSF，，建立M-CSF核内稳定表达细胞系，探讨核内M-CSF对NIH3T3细胞运动和细胞骨架的影响。方法：采取PCR的方法扩增人M-CSF活性片段，将其插入真核表达载体pEGFP-C1中，PCR、酶切分析及测序鉴定筛选阳性重组体pEGFP／M-CSF，经脂质体瞬时转染NIH3T3细胞，用荧光显微镜观察GFP-M-CSF融和蛋白在NIH3T3细胞内的表达分布，以确定在未加信号肽的条件下M-CSF在NIH3T3细胞中的定位。然后将M-CSF片段插入核内真核表达载体pCMV／myc／nuc，强制性把M-CSF引入细胞核内，通过PCR、酶切分析及测序鉴定筛选阳性重组体pCMV／M-CSF，脂质体介导转染NIH3T3细胞，经G418筛选并扩增阳性克隆后，用RT-PCR、免疫细胞化学及western blot鉴定其在真核细胞中的表达及定位分布，用细胞划痕实验和考马斯亮蓝染色细胞微丝测定M-CSF进入细胞核后对细胞运动能力及细胞骨架的影响。结果：PCR扩增和限制性双酶切重组体pEGFP／M-CSF和pCMV／M-CSF后，琼脂糖电泳结果显示插入片段为1400bp左右，与预期M-CSF分子大小相当；DNA测序分析表明插入质粒的M-CSF无读码框移位，并与来源序列一致。荧光显微镜下见到GFP-M-CSF融和蛋白表达定位于NIH3T3细胞质和细胞核。RT-PCR和Westorn blot结果显示转染pCMV／M-CSF的NIH3T3细胞能稳定表达M-CSF mRNA与M-CSF蛋白；免疫细胞化学结果显示表达的M-CSF定位于NIH3T3细胞核。细胞划痕实验显示转染pCMV／M-CSF的NIH3T3细胞有较强的运动能力，考马斯亮蓝染色结果显示转染pCMV／M-CSF的NIH3T3细
[Abstract]:Aim: to construct eukaryotic expression vector pEGFP / M-CSF of GFP-M-CSF fusion protein, analyze the localization and distribution of M-CSF in NIH3T3 cells, construct M-CSF expression vector pCMV / M-CSF in nucleus, and establish a stable expression cell line of M-CSF nucleus. To investigate the effects of nuclear M-CSF on NIH3T3 cell motility and cytoskeleton. Methods: the active fragment of human M-CSF was amplified by PCR and inserted into the eukaryotic expression vector pEGFP-C1. The positive recombinant pEGFP / M-CSF was screened by restriction endonuclease analysis and sequencing. NIH3T3 cells were transiently transfected with liposome. The expression and distribution of GFP-M-CSF fusion protein in NIH3T3 cells were observed by fluorescence microscope to determine the location of M-CSF in NIH3T3 cells without signal peptide. Then the M-CSF fragment was inserted into the eukaryotic expression vector pCMV / myc / nuclear, and M-CSF was forcibly introduced into the nucleus. NIH3T3 cells were transfected by liposome mediated by pCMV / M-CSF by PCR, restriction endonuclease analysis and sequencing. After G418 screening and amplification of positive clones, the expression and localization of pCMV / M-CSF in eukaryotic cells were identified by RT-PCR, immunocytochemistry and western blot. The effects of M-CSF on cell motility and cytoskeleton were measured by cell scratch test and Coomassie brilliant blue staining. Results after PCR amplification and restriction digesting the recombinant pEGFP/M-CSF and pCMV/M-CSF, the agarose electrophoresis results showed that the inserted fragment was about 1400bp, and the sequence analysis showed that the M-CSF of the inserted plasmid was translocated without reading frame, which was equivalent to the expected size of M-CSF. The results of RT-PCR and Westorn blot showed that the NIH3T3 cells transfected with pCMV/M-CSF could stably express M-CSF mRNA and M-CSF protein. The results of immunocytochemistry showed that the expressed M-CSF was located in the nucleus of NIH3T3. The cell scratch assay showed that the NIH3T3 cells transfected with pCMV/M-CSF had strong motor ability. Coomassie brilliant blue staining showed that the NIH3T3 transfected with pCMV/M-CSF was fine.
【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】：R346

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