细胞质M-CSF对NIH 3T3细胞增殖和运动的影响

发布时间：2018-03-05 04:17

本文选题：M-CSF　切入点：NIH　出处：《南华大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的：建立胞质内稳定表达M-CSF的细胞系；探讨定位在胞质内的M-CSF对细胞增殖和运动的影响。方法：PCR分别扩增人M-CSF的胞外活性区和功能区，连接到胞浆定位载体pCMV／cyto／myc质粒，，得重组质粒pCMV／cyto／myc-h-M-CSF，再将重组质粒稳定转染NIH 3T3细胞，得到分别稳定表达M-CSF的胞外活性区和功能区的细胞株。免疫细胞化学验证M-CSF蛋白的定位，细胞生长曲线和致伤愈合实验显示外源的M-CSF对NIH3T3细胞增殖和运动的影响。考马斯亮兰对细胞骨架进行染色。结果：重组基因的插入片段大小分别与M-CSF的胞外区和功能区相符，重组的M-CSF片段无阅读框移位和突变，绿色荧光蛋白仅定位在NIH3T3细胞质内，表明成功构建胞浆定位表达载体pCMV／cyto／myc-M-CSF-a和pCMV／cyto／myc-M-CSF-d；分别将pCMV／cyto／myc-M-CSF-a和pCMV／cyto／myc-M-CSF-d质粒转染NIH3T3细胞，并经G418筛选后，稳定表达M-CSF胞外区和功能区的NIH 3T3细胞株其倍增时间均短于对照细胞；胞质内M-CSF诱导NIH3T3细胞微丝重构和细胞迁移；M-CSF N末端149个氨基酸残基组成的功能区其促NIH3T3细胞增殖和运动活性与M-CSF的胞外区基本相同。结论：建立了一个细胞质稳定表达M-CSF的细胞系；细胞质M-CSF能诱导NIH 3T3细胞增殖，增强细胞运动能力；M-CSF蛋白的功能区诱导细胞增殖与迁移的生物学活性与M-CSF胞外区的活性基本相同。
[Abstract]:Aim: to establish a cell line with stable expression of M-CSF in cytoplasm and to investigate the effect of M-CSF localized in cytoplasm on cell proliferation and motility. Methods the extracellular active and functional regions of human M-CSF were amplified by 10: PCR and ligated to the plasmids of cytoplasmic localization vector pCMV/cyto/myc. The recombinant plasmid pCMV / cyto- myc-h-M-CSF was then stably transfected into NIH. 3T3 cell lines expressing the extracellular active and functional regions of M-CSF were obtained, respectively. The localization of M-CSF protein was confirmed by immunocytochemistry. The cell growth curve and wound healing test showed that exogenous M-CSF had an effect on the proliferation and movement of NIH3T3 cells. Coomassie brilliant blue stained the cytoskeleton. Results: the insert size of the recombinant gene was consistent with the extracellular and functional regions of M-CSF respectively. The recombinant M-CSF fragment had no reading frame translocation or mutation, and the green fluorescent protein was located only in the cytoplasm of NIH3T3. The results showed that the cytoplasmic localization expression vectors pCMV/cyto/myc-M-CSF-a and pCMVr-myc-M-CSF-dwere successfully constructed, and the pCMV/cyto/myc-M-CSF-a and pCMV/cyto/myc-M-CSF-d plasmids were transfected into NIH3T3 cells respectively. After G418 screening, the doubling time of NIH3T3 cells expressing the extracellular and functional regions of M-CSF was shorter than that of the control cells. Cytoplasmic M-CSF induces NIH3T3 cell microfilament remodeling and cell migration. The functional region composed of 149 amino acid residues at the N-terminal of M-CSF can promote the proliferation and motor activity of NIH3T3 cells, which is basically the same as the extracellular domain of M-CSF. Conclusion: a cell line with stable expression of M-CSF in cytoplasm was established, and cytoplasm M-CSF could induce the proliferation of NIH 3T3 cells. The biological activity of cell proliferation and migration induced by the functional region of M-CSF protein was basically the same as that of the extracellular domain of M-CSF.
【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】：R363

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