| Background and objectives of the study:Neural stem cell is a hotspot topic in the field of neuroscience in the past few years. The self-renewal potential and multipotentiality of neural stem cells have brought new hope for the treatment of a variety of nervous system diseases such as Parkinson's disease. However, a key problem exists: the induction mechanism of the directed differentiation of neural stem cells into the neurons needed is still unclear. How to realize the directed differentiation induction of neural stem cells is the core issue of neural development research. The current study shows that there are two mechanisms of the directed differentiation determination of neural stem cells: one is the epigenetic mechanism of cell development and differentiation, the other is the regulation of external signals majored by various cytokines, namely the micro-environment. In which the regulation of key genes plays a crucial role in the proliferation and differentiation of neural stem cells, which determines the differentiation direction of neural stem cells into specific neurons. At the moment there are mainly two ways to study this topic: (1) in vivo study, the stimulating effects of the genes on the differentiation direction of neural stem cells are studied via gene transduction and knock-out; (2) in vitro study, the effects of genes under induced stimulation on the proliferation and differentiation of neural stem cells via induction of specific genes using various cytokines. However, the gene regulation mechanism involved is still not clear so far. Therefore, the study on the gene regulation mechanism of neural stem cells is very important.Anterior subventricular zone (SVZa) is known as one of the regions with concentrated neural stem cells. From the embryo stage to the adult stage, the neural stem cells in this zone continuously move to the Olfactory bulb (OB) along the Rostral migratory stream (RSM) under the control of the in vivo dorsal - ventral signal and finally differentiate at OB into interneurons with phenotype of OB, including dopaminergic neurons. Besides the self-renewal potential and multipotentiality possessed by neural stem cells in general, SVZa neural stem cells also have other properties: (1) they possess the potential to develop into neurons since their birth; (2) they have fixed migration pathway, they are highly localized and they need to migrate over long distance; (3) they keep the proliferation state and the neuron differentiation potential and do not further differentiate all the time during the migration. Therefore, SVZa neural stem cells have become a good model for studying the regulation mechanism of proliferation, migration and differentiation in neural stem cells and become the emphasis in the studies on neural stem cells.During the migration to OB and differentiation, SVZa neural stem cells require the complicated environmental signals in vivo and in vitro for regulation, including DLX5 and Bone morphogenetic protein-2 (BMP-2) etc. Studies have shown that DLX5 gene in the DLX (distall-less homeobox) gene family is closely related to the development of OB interneurons. It has been found previously using gene knock-out rats that DLX5 gene plays an important role in the differentiation of SVZa neural stem cells into OB dopaminergic interneurons and is an essential gene for neurogenesis after birth. Although in vivo study has verified that DLX5 gene is an essential gene for OB dopaminergic interneurons after birth, the detailed regulation mechanism of DLX5 in neural stem cells differentiation is still not clear so far. Furthermore, since the DLX5 gene knock-out rats died in the perinatal stage, further research focused on the in vitro studies.As an important member of the Bone morphogenetic proteins (BMPs) family, BMP-2 is a very important cytokine in the micro-environment where the stem cells are localized. It was found that it had different effects on neural stem cells at different development stages and different sites. It can promote the differentiation of neural stem cells not only into neurons but also into astrocytes. In fetal rat SVZa, BMP-2 was able to promote the differentiation of neural stem cells into neurons and significantly promote its differentiation into dopaminergic neurons. Studies have proven that DLX5 gene is an essential gene for the differentiation of OB dopaminergic interneurons, so what are the genes that can regulate DLX5 to help the differentiation of SVZa neural stem cells into dopaminergic neurons? In combination with previous studies, it was found that BMP-2 and DLX5 played an important role in the differentiation of SVZa neural stem cells into OB dopaminergic neurons; and that BMP-2 significantly promoted the expression of bone tissue DLX5. Then during the differentiation of SVZa neural stem cells into dopaminergic neurons, does BMP-2 act as an upstream regulator of DLX5 and promote the differentiation of SVZa neural stem cells into dopaminergic neurons via regulating DLX5 expression? Therefore, first SVZa neural stem cells were isolated and cultivated in this study to serve as the research object, the effects of BMP-2 and its antagonist Noggin on the differentiation of SVZa neural stem cells into dopaminergic neurons were checked and the expression level of DLX5 during the whole process was tested with immunofluorescence staining and RT-PCR, in order to prove that BMP-2 induced the differentiation of SVZa neural stem cells into dopaminergic neurons via the DLX5 pathway. Hopefully this study can provide some experimental data for the directed differentiation research on neural stem cells.Methods:1,SVZa neural stem cells were isolated and cultivated using cell culture technique and the neurospheres obtained were subjected to cellular immunologic Nestin identification. After differentiation, the neurospheres were analyzed for NSE, GFAP, Oligo-2 with cellular immunologic identification, to confirm its stem cell properties and multipotentiality.2,The SVZa neural stem cells obtained from the same batch of in vitro culture were induced with different factors and divided into different groups: control group, BMP-2 group, BMP-2+Noggin group and Noggin group. The expression of DLX5 protein and DLX5 mRNA of the different treatment groups was detected using immunofluorescence staining and RT-PCR, respectively.3,The SVZa neural stem cells obtained from the same batch of in vitro culture were induced with different factors and divided into different groups: control group, BMP-2 group, BMP-2+Noggin group and Noggin group. The percentage of SVZa neural stem cells differentiated into dopaminergic neurons in different treatment groups was detected with immunofluorescence staining and flow cytometry.Results:1. In the serum-free culture in vitro, a large amount of neurospheres in suspended growth were obtained, which was positive in Nestin immunofluorescence staining and the NSE, GFAP, Oligo-2 cellular immunologic identification was also positive.2. After induced differentiation for 72 hours, the control group, BMP-2 group, BMP-2+Noggin group and Noggin group were detected with immunofluorescence staining and RT-PCR, respectively. The results showed that the DLX5 expression was significantly enhanced in the BMP-2 group, whereas the DLX5 expression had no significant change in the control group, BMP-2 +Noggin group and Noggin group; the expression level of DLX5 mRNA in the BMP-2 group was also significantly higher than the control group, p<0.05, the difference was statistically significant; whereas the comparison among the control group, BMP-2 +Noggin group and Noggin group showed no significant difference in the expression level of DLX5 mRNA, p>0.05.3. After induced differentiation for 72 hours, the control group, BMP-2 group, BMP-2+Noggin group and Noggin group were detected with immunofluorescence staining and flow cytometry, respectively. The results showed that there were more TH+ cells seen in the BMP-2 group, whereas there were significantly fewer TH+ cells found in the control group, BMP-2 +Noggin group and Noggin group; The results of flow cytometry also indicated that the percentage of differentiated TH+ neurons was significantly higher in the BMP-2 group than in the control group, p<0.05, the difference was statistically significant; whereas the comparison among the control group, BMP-2 +Noggin group and Noggin group showed no significant difference in the percentage of differentiated TH+ neurons, p>0.05.Conclusion:1,Neural stem cells were successfully isolated from the SVZa brain tissue of Wistar rat at the 15th day of pregnancy, which had continued proliferation and self-renewal capacity and were able to differentiate into neurons, astrocytes and oligodendrocytes. 2,BMP-2 significantly promoted the expression of DLX5 protein and DLX5 mRNA in SVZa neural stem cells and this effect could be antagonized by its antagonist Noggin.3,BMP-2 significantly promoted the differentiation of SVZa neural stem cells into dopaminergic neurons, and this effect could also be antagonized by its antagonist Noggin. In SVZa, BMP-2 might promote the differentiation of neural stem cells into TH+ neurons via up-regulating the expression of DLX5. |
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