Pax3对神经细胞中的转录调控作用的实验研究

The Study on the Roles of Pax3 in the Transcriptional Regulation of Nerve Cells

目的:探讨Pax3的过表达对Neuro-2a细胞中转录本的表达影响,初步分析Pax3对Neuro-2a细胞可能的转录调控作用。方法:反复冻融裂解法获取Pax3过表达腺病毒后将神经瘤母细胞系Neuro-2a传代培养,而后将Pax3过表达腺病毒和传代培养后的Neuro-2a细胞加入到同一培养皿中,蛋白质免疫印迹(Western blot)检测过表达Pax3蛋白的Neuro-2a细胞(Pax3过表达组)和对照组(NC组)Neuro-2a细胞的Pax3蛋白表达水平,实时荧光定量PCR(qRT-PCR)法检测Pax3过表达组和NC组Neuro-2a细胞的Pax3mRNA水平,Trizol法提取Pax3过表达组和NC组Neuro-2a细胞的总RNA,然后进行全转录本测序,最后将选出的有差异性的基因使用实时荧光定量PCR(qRT-PCR)验证。结果:与NC组相比,Pax3过表达组的Pax3蛋白和Pax3mRNA表达水平明显升高(P<0.05);Pax3过表达组中发现了1045个基因表达上调,1313个基因表达下调。通过qRT-PCR验证发现在Pax3过表达组中Nppb和Chrna5表达水平上升(P<0.05),Arhgap5、Rock1、Rif1、Brca2、Prkg2和Stag2表达水平下降(P<0.05)。结论:Pax3过表达腺病毒感染Neuro-2a细胞后,其蛋白和m RNA表达水平均升高,Rock1、Rif1和Stag2可能作为Pax3的下游靶点参与调控Neuro-2a细胞周期和干细胞特性。

Objective: To investigate the transcriptional differences in Neuro-2 a cells overexpressing Pax3 and analyze the possible transcriptional regulation of Pax3 on Neuro-2 a cells. Methods: The overexpression adenovirus of Pax3 was obtained by repeated freeze-thaw method, and neuroblastoma cell line Neuro-2 a was subcultured, Then, Pax3 overexpression adenovirus and subcultured Neuro-2 a cells were added into the same culture dish,Western blot was used to detect the expression of Pax3 protein in Neuro-2 a cells(Pax3 overexpressed group) and control group(NC group), Real time quantitative PCR(qRT-PCR) was used to detect the expression of Pax3 m RNA in Neuro-2 a cells of Pax3 overexpressed group and NC group. Total RNA of Pax3 overexpressed group and NC group that overexpressed Pax3 protein were extracted by Trizol. and then the whole transcript was sequenced, the selected genes were verified by qRT-PCR in the end. Results: Compared with NC group, The expression levels of Pax3 protein and Pax3 m RNA in Pax3 overexpressed group were significantly increased(P<0.05);the expression levels of 1045 genes were upregulated and 1313 genes were downregulated in Pax3-overexpressed group. The results of qRT-PCR verified the expression changes of Nppb and Chrna5 increased(P<0.05), while the expression changes of Arhgap5, Rock1, Rif1, Brca2, Prkg2 and Stag2 decreased in Pax3 overexpressed group(P<0.05). Conclusion:Rock1, Rif1 and stag2 may be downstream targets of Pax3 to regulate cell cycle and stem cell characteristics of Neuro-2 a.