摘要
目的研究D lx5 mRNA在培养的室管膜前下区(anterior subventricu lor zone,SVZa)神经干细胞(NSCs)中的表达规律及其对SVZa NSCs方向分化的影响。方法提取每一代培养的SVZa NSCs的总RNA,通过逆转录-聚合酶链反应(RT-PCR)检测D lx5及Er81 mRNA表达的情况;将重组质粒pEGFP-mD lx5用电穿孔的方法转染不表达D lx5 mRNA代次的NSCs,再检测D lx5 mRNA的表达情况,并将转染细胞分化后进行免疫荧光染色,分析基因转染细胞与神经元分化的关系;另外,还用100 ng/m l的骨形态发生蛋白-2(BMP-2)处理不表达D lx5 mRNA的NSCs,24 h后检测D lx5 mRNA的表达情况。最后将不同D lx5 mRNA表达情况的NSCs分化后用流式细胞仪检测神经元分化的比例。结果细胞传代至第4代时D lx5 mRNA表达消失,相应地,其神经元分化比例为(8.34±0.36)%,比表达D lx5 mRNA的第3代NSCs的(19.16±0.75)%明显下降;用D lx5基因转染该代NSCs后,外源转染基因能够使内源性D lx5 mRNA恢复表达,神经元分化比例也明显升高,为(35.66±0.58)%,免疫荧光染色也证实基因转染细胞分化成了神经元;而BMP-2处理也能诱导D lx5 mRNA重新表达,神经元分化比例也有一定程度上升,与第3代NSCs的相似,为(22.27±0.12)%。除第3代NSCs分化组与BMP-2诱导组神经元分化比例差异无统计学意义外,其余各组间两两比较差异均有统计学意义(P<0.05)。结论随着传代次数的增加,到第4代时,SVZa NSCs D lx5 mRNA表达消失,而外源性D lx5基因转染及BMP-2可以诱导其重新表达;D lx5基因能使新生大鼠来源的SVZa NSCs向神经元方向分化。
Objective To study the expression of Dlx5 mRNA in in vitro expanded neural stem cells （NSCs） derived from the anterior subventricular zone （SVZa）of the neonatal rats and its effect on SVZa NSCs differentiating into neurons. Methods Total RNA of every passage of NSCs was extracted and by transcription-polymerase chain reaction （ RT-PCR）, to understand mRNA expressions of Dlx5 and Er81. The recombinant pEGFP-mDlx5 plasmids were transfected to SVZa NSCs by electroporation to analyze the expression of Dlx5 mRNA. The transfected cells were differentiated for immunofluorescence stain to understand the relation between gene-transfected cells with neuron differentiation. Furthermore, NSCs without expressing Dlx5 mRNA was treated with bone morphogenetic protein-2 （BMP-2） at concentration of 100 ng/ml and, after 24 hours, to detect expression of Dlx5 mRNA. Finally, the differentiation proportion of the neurons was detected by flow cytometry after NSCs with various different expression of Dlx5 mRNA was differentiated. Results No Dlx5 mRNA could be detected at the fourth passage of NSCs. Correspondingly, the differentiation proportion of neuron was decreased from （19.16±0.75 ） % to （8.34 ± 0.36） % at the third passage. However, expression of endogenous Dlx5 mRNA could be recovered by exogenous gene after Dlx5 was used to transfect the third passage of NSCs, with neuron increased to （35.66±0.58 ） %. The immunofluorescence staining showed that the cells transfected with Dlx5 gene were differentiated into neuron. Treatment with BMP-2 could induce re-expression of Dlx5 mRNA, with certain degree of proportion increase of neuron differentiation, ie, （22.27±0.12）%, which was similar to the third passage of NSCs. As for portion of neuron differentiation, there was significant difference between other groups by analysis of variance （ P 〈 0.05 ）, except for comparison between the third passage ceils group and the BMP-2 treated group. Conclusions Expression of Dlx5 mRNA disappears at the fourth passage of SVZa NSCs but can be recovered by exogenous gene transfection and BMP-2. In the meantime, Dlx5 gene can direct in vitro expansion of SVZa NSCs differentiating into neurons.
出处
《中华创伤杂志》
CAS
CSCD
北大核心
2006年第5期367-371,共5页
Chinese Journal of Trauma
基金
国家自然科学基金重点资助项目(30130110)
