转录因子Phox2对SK-N-BE(2)C细胞中酪氨酸羟化酶表达的影响  

Effect of transcription factors Phox2 on expression of tyrosine hydroxylase in SK-N-BE(2)C cells

作　　者：范雁[1] 杜向东[1] 杨勇[1] 殷光中[1] 朱梦漾 

机构地区：[1]苏州大学附属广济医院,苏州215008 [2]美国东田纳西州立大学奎林医学院

出　　处：《神经解剖学杂志》2014年第5期562-568,共7页Chinese Journal of Neuroanatomy

基　　金：苏州市科技局应用基础研究项目(SYS201372);美国国立卫生院R21(NS052818)

摘　　要：目的：本研究通过观察转录因子Phox2（Phox2a和Phox2b）对成神经细胞瘤细胞株SK-N-BE（2）C细胞中酪氨酸羟化酶（tyrosine hydroxylase，TH）表达的影响来证实Phox2对去甲肾上腺素能表型的决定性作用。方法：将携带人类Phox2a或2b cDNAs的质粒、对Phox2a或Phox2b特异的shRNAs质粒分别转染培养的SK-N-BE（2）C细胞，转染后2-3d收集各组细胞，RT-PCR法检测Phox2a、Phox2b、TH等在mRNA水平的表达；Western Blot法检测各指标在蛋白水平的表达。结果：Phox2的过表达导致TH在mRNA和蛋白水平的表达显著增加；Phox2的沉默表达则使TH在mRNA和蛋白水平的表达明显降低，差异有统计学意义（P〈0．01或P〈0．05）。结论：Phox2对TH的表达具有调节作用，从而在细胞水平证实了Phox2对神经系统去甲肾上腺素能系统表型的决定性作用，该结果将为治疗与去甲肾上腺素能机能失调相关的老年痴呆、抑郁症等探索新途径。Objective： To investigate the effect of two transcription factors Phox2 （Phox2a and Phox2b）on expression of tyrosine hydroxylase in SK-N-BE（2）C neuroblastoma cells and then to further verify the decisive role of Phox2 to the no- radrenergic phenotype. Methods： The human neuroblastoma cell line SK-N-BE （2）C cells were grown to about 90% con- fluence and separately transfected with pCMV6-XL5 plasmid DNA carrying cDNAs of human Phox2a or Phox2b, shRNAs specific to Phox2a or Phox2b genes. Cells were harvested 2-3 days after transfection for different measurements. The mR- NA levels of Phox2a or Phox2b, and TH were measured by RT-PCR; the protein levels were measured with Western Blot. Results： The over expression of Phox2 significantly increased TH mRNA and protein levels, which didn＇t show a concen- tration-dependent manner; shut-down of endogenous Phox2 reduced mRNA and protein levels of TH （P 〈 0.01 or P 〈 0.05）. Conclusion： The present findings verify the determinant role of Phox2a and Phox2b on the expression of TH in vitro. Further clarifying the regulatory role of these two transcription factors on key proteins of the noradrenergic system may open a new avenue for therapeutics of aging-caused dysfunction of the noradrenergic system.

关 键 词：Phox2 成神经细胞瘤细胞株 酪氨酸羟化酶 去甲肾上腺素能表型 SK-N-BE(2)C细胞 

分 类 号：R3411[医药卫生—基础医学]