【摘要】目的 观察IκBα突变型基因对永生化神经前体细胞株（INPCs）增殖、分化及部分炎性细胞因子分泌的影响。方法 在已经建立的转染pcDNA3.1及转染pcDNA3.1/IκBαM的INPCs的基础上，用MTT法绘制两细胞株的生长曲线，用流式细胞仪检测其增殖指数，以5％小牛血清诱导分化后，采用western blot检测两株细胞的分化情况，同时用realtime RT-PCR比较TNF-α、IL-1β和IL-6在两种细胞株中的表达。 结果 生长曲线及流式细胞仪测得的细胞增殖指数提示：转染pcDNA3.1/IκBαM INPCs的增殖速度较转染pcDNA3.1的INPCs明显降低。经血清诱导分化后，两者均大量表达胶原纤维酸性蛋白；转pcDNA3.1/IκBαM的INPCs较转pcDNA3.1的INPCs IL-1β和IL-6的表达减低，但TNF-α表达变化无显著性。结论 IκBα突变型基因可减慢INPCs的增殖，并减少部分炎性细胞因子的表达，但对INPCs的分化无显著影响。
【关键词】干细胞； NF-κB抑制因子α；细胞增殖；细胞分化；细胞因子

Effect of mutated IκBα gene on the biological characteristics of immortalized neural progenitor cell strain
ZHU Chang, LIU Zhi-heng, ZHANG Chuan-han, et al. Department of Anesthesiology，Tongji Hospital of Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China

【Abstract】 Objective To observe the effect of mutated IκBα gene on the proliferation，differentiation and cytokine secretion of immortalized neural progenitor cell strain(INPCs). Methods After the establishment of mutated IκBα gene transfected INPCs and blank plasmid transfected INPCs, MTT staining and flow cytometry were used to draw the growth curve and measure the cell proliferation index. Western blot was carried out to observe the differentiation of the two cell strains after 5% blood serum induced differentiation. The expression of TNF-α、IL-1β and IL-6 was measured by realtime RT-PCR. Results It was establish免费论文网 【http://www.51lunwen.net】ed by growth curve and cell proliferation index that the proliferation was slowed down in mutated IκBα gene transfected INPCs compared with blank plasmid transfected INPCs. After blood serum induced differentiation，glial fibrillary acidic protein was highly expressed in the two cell strains. There was no difference in the expression of TNF-α between mutated IκBα gene transfected INPCs and blank plasmid transfected INPCs. The expression of IL-1β and IL-6 was reduced in mutated IκBα gene transfected INPCs. Conclusion Mutated IκBα gene can slow down the proliferation of INPCs, reduce the expression of some cytokines, but didn’t influence the differentiation of INPCs.
【Key words】Stem cells；NF-kappaB inhibitor alpha；Cell proliferation ；Cell differentiation； Cytokines
本实验室成功构建了SV40Tag永生化的大鼠神经前体细胞株（INPCs）[1]，并在此基础上构建了转IκBα突变型基因永生化神经前体细胞株。作为细胞移植治疗的细胞来源和外源基因的载体，INPCs的生物学特性在转入外源基因后有何改变，是进一步研究首先应关注的问题。本研究拟通过比较转IκBα突变型基因及空白对照载体的INPCs在增殖、分化以及细胞因子分泌功能上的差异，为进一步将该转基因细胞用于细胞移植治疗脑缺血等中枢神经系统疾病奠定基础。
材料与方法
材料 转染pcDNA3.1及pcDNA3.1/IκBαM的INPCs由本实验室构建，DMEM/F12培养基、B27无血清培养添加剂（Gibco公司，美国）；碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)（Pepro Tech公司，英国）； MTT、碘化丙啶（PI）（Sigma公司，美国）；抗β-actin抗体、抗胶原纤维酸性蛋白（GFAP）抗体、抗微管相关蛋白2（MAP2）抗体（Neomarkers公司，美国）； Trizol试剂（上海华舜公司），SYBR ExScriptTM RT-PCR Kit (Takara，日本)。
细胞培养及传代 转染pcDNA3.1或pcDNA3.1/IκBαM的INPCs在含2%B27、20ng/ml bFGF、20ng/ml EGF、0.3μg/ml嘌呤霉素的DMEM/F12培养基中，置37℃、5%CO2培养箱培养，0.25％胰酶消化传代。
细胞生长曲线的绘制 将转染pcDNA3.1及pcDNA3.1/IκBαM的INPCs以每孔5×103的密度接种于96孔板，共种8板，每板每株细胞接种12孔， 本文来自：免费论文网 [http://www.51lunwen.net]