RNAi技术在神经干细胞相关基因功能研究中的应用

目的:采用RNA干涉(RNA interference,RNAi)技术研究神经干细胞相关基因-AF116909在神经干细胞中的作用。方法:构建含AF116909目的基因特异序列的发夹结构RNA(small hairpin RNA,shRNA)表达载体,转染到神经干细胞(NSCs)后,观察神经干细胞的形态,并进行RT-PCR检测mRNA表达。结果:经观察,试验组分化明显加强,从3d的(12±1.5)%分化率增加到11d的(80±3.1)%,而对照组几乎无分化现象。RT-PCR检测后,发现试验组中AF116909基因mRNA的表达量同对照组相比明显减少。结论:该方法实现了AF116909基因在NSCs中的有效抑制,并表明,抑制AF116909基因的表达,提高了NSCs的分化率,反应该基因具有抑制NSCs分化的功能。

RNAi techrique and Its application in gene function research of NSCs

Objective:To explore the function of AF116909 gene in neural stem cells by RNAi technology.Methods:we constructed shRNA expression vector which was formed by a pair of DNA sequences corresponding to AF116909 ligate with pSilec Circle vector containing U6 promotor of RNA Pol Ⅲ.After the vector is transfected in to NSCs,observation and detection by were carried RT-PCR.Result:The differentiation of NSCs was strengthened and the differentiation ratio was increased to(80±3.1)% in the elecenth day from(12±1.5)%in the third day.After RT-PCR,The expression amount of mRNA of AF116909 in test group was much less than that in control group.Conclusion:These findings show that transfected as well as endogenous genes can be efficiently inhibited and AF116909 gene has the function of inhibiting the differentiation of NSCs.