摘要
随着神经干细胞(neural stem cells, NSCs)研究的日渐深入,室管膜前下区(anterior subventricular zone, SVZa)作为目前公认的哺乳动物中枢神经系统神经干细胞比较集中的一个重要区域,越来越受到人们的关注。SVZa来源的NSCs不仅具备所有神经干细胞的共性,而且此处神经干细胞产生后,将沿着一条特定的迁移通道――喙侧迁移流(rostral migratory stream, RMS)朝嗅脑(球)迁移,在迁移过程中始终保持增殖状态和神经元前体细胞的特征而不进一步分化,到达其迁移终点后才进一步分化为成熟的神经元,成为嗅脑球中间神经终生更新的来源,这也是SVZa神经干细胞有别于其他来源神经干细胞的主要特征。然而,在SVZa神经干细胞增殖、迁移、分化的一系列过程中,不但制约于自身的遗传基因,而且受到细胞所处微环境的影响,而微环境中多种细胞因子参与了调控。最新的研究显示BMPs、Mash1、Pax6和Wnt等多种细胞因子和分子涉及了这些复杂过程,但SVZa神经干细胞增殖、迁移和分化的具体调控机制目前仍不清楚。
骨形成蛋白(Bone Morphogenitic Proteins,BMPs)是转化生长因子-β(Transforming Growth Factor-β,TGF-β)超家族成员,是一组具有多种功能的细胞因子,最初发现他们能够在体外诱导骨和软骨的形成而得名。目前在哺乳动物体内克隆和表达了二十多种BMPs成员,包括BMP-2、BMP-3、BMP-4、BMP-5等等,他们结构相似,功能相关,广泛分布于机体各处。BMPs通过与细胞膜上的特异性受体(BMPRs)结合,将信号传导到胞内,促进胞浆内Smads家族成员的磷酸化,而后者形成功能复合物进入核内,进而调控下游目标基因的转录。BMP-2是BMPs成员中被研究得较为广泛的一个因子,研究表明BMP-2不但对骨和软骨的发生有促进作用,而且参与神经前体细胞的分化成熟与凋亡的调节,对神经系统的发生发育起着重要调控作用。研究发现,BMP-2及其受体表达在神经系统发育的各个阶段,对神经干细胞的增殖与定向分化有一定影响,同时也有研究表明BMP-2浓度依赖性地诱导神经前体细胞分化为神经元。
国家自然科学基金重点项目:No.30130110
* 国家自然科学基金青年项目:No.30300463
而相关研究证实,BMP-2功能的实现依赖于其受体信号途径,其中一条途径是BMP-2-BMPRs-Smads-靶基因,BMP-2通过该信号途径直接影响神经干细胞的分化。然而,对于SVZa神经干细胞,其由经RMS迁移至嗅球并进行最终分化,这个特殊而又复杂的迁移、分化过程中,BMP-2可能起到怎样的调节作用,其具体机制又是如何,目前还未见相关报道。虽然最近研究显示一定浓度的人重组BMP-2(rhBMP-2)单独作用于体外培养的SVZa神经干细胞,能有效诱导神经干细胞分化为神经元。但其具体的作用机制还不清楚。
在本实验中,我们首先用抗nestin、NF、GFAP和BMP-2等进行免疫荧光染色,观察在发育不同阶段的大鼠脑内SVZa神经干细胞喙侧迁移流通路上BMP-2的分布情况,同时结合RT-PCR技术,对BMP-2在大鼠脑神经干细胞喙侧迁移流中的发育学表达模式进行深入的分析,得到如下结果:
1.在大鼠发育的各个阶段,NSCs自SVZa向嗅球迁移,形成明显的神经干细胞迁移流;nestin阳性细胞在SVZa区及其周围,RMS迁移流主干和OB中央有较强表达,但随着大鼠的发育成熟,各部位nestin阳性细胞渐减;
2.NF和GFAP在迁移流上表达较弱,主要表达在侧脑室,SVZa区周围,在RMS几乎没有GFAP的表达,NF的表达也很低,但在OB周围NF和GFAP均有较强表达;
3.BMP-2在喙侧迁移流的表达模式:①时间差异:在大鼠SVZa区、RMS主干部分和嗅球三个部位,BMP-2的表达水平在胚胎14d明显高于出生后1d、7d和成年三个时间点,差异较为显著,在出生后0d和7d这两个时相点上,BMP-2的表达水平相似,而成年BMP-2水平为最低。②空间差异:胚胎14天,BMP-2在SVZa表达水平最高,嗅球次之,而其他三个时相点嗅球表达较高,在SVZa区的表达居次,在RMS表达量始终最低。
以上结果证实了喙侧迁移流主要由SVZa来源的神经干细胞组成,而BMP-2在迁移流通路上的表达模式提示其不但参与了SVZa神经干细胞的增殖和迁移,而且对其分化可能也有重要的影响,为了进一步证实BMP-2对SVZa神经干细胞分化的作用,我们体外培养了SVZa神经干细胞,用不同浓度的BMP-2蛋白对体外培养的SVZa神经干细胞进行诱导,初步探讨了BMP-2对SVZa神经干细胞的分化的影响,实验结果如下:
1.在无血清等其他因子条件下,BMP-2可以有效刺激SVZa神经干细胞分化,与自然条件相比,有加速神经干细胞分化趋势。BMP-2抑制SVZa神经干细胞表达nestin,促进其分化成熟;
2.在5-25ng/ml浓度范围内,BMP-2均不同程度的促进SVZa神经干细胞向神经元方向分化,并且有轻微抑制SVZa神经干细胞向胶质细胞方向分化的作用,其中10ng/ml是促进SVZa神经干细胞向神经元分化的最佳浓度;
3.在5-25ng/ml浓度的BMP-2有利于SVZa神经干细胞分化为TH阳性神经元,其中10ng/ml浓度效果最佳,但TH阳性神经元的比例整体较低。
上述结果证实了BMP-2浓度依赖性地对体外培养的SVZa神经干细胞的定向分化具有一定的影响,为了明确BMP-2对SVZa神经干细胞分化调控的可能分子机制,我们拟用实验中的最佳作用浓度――10ng/ml BMP-2刺激下,在不同的时相点,观察在分化的不同时期BMP-2信号途径中胞浆内S
With the lucubrating studies on neural stem cells (NSCs), the anterior subventricular zone (SVZa) as a special area where NSCs are concentrated has been focused by more and more reseachers. NSCs derived from SVZa not only possess the characteristics of NSCs, but also have their own features: 1) they will migrate form SVZa to olfactory bulb (OB) along an especial routeway- the rostral migratory stream (RMS) after they are produced; 2) they will maintain proliferating and the characters of neural progenitor; 3) they differentiate into maturated neurons while migrating to destination and become the renewing resource of OB interneurons in the whole life of mammal animals, which is the unique character of SVZa NSCs differed from other NSCs. However, the development of SVZa NSCs are affected not only by inherit genes, but also by the extracellular signals. Recently, there were increasing evidences that many extracellular signals regulate the proliferation and differentiation of NSCs, such as mitogenic factors (EGF), transcription factors (BMPs, Mash1, Pax6, and Wnt) and so on.
Increasing evidences suggested that the fate of neural progenitors is regulated by members of BMPs. BMPs belong to transforming growth factor-β (TGF-β) superfamily, which were originally identified and characterized by their novel ability to induce cartilage and bone formation in ectopic extraskeletal site in vivo. Recently, several members of this family have been isolated, cloned, and expressed as recombinant protein in mammals, include BMP-2, BMP-3, BMP-4 and BMP-5 and so on. BMPs exert their cellular effects by binding to transmembrane receptors that possess serine/threonine kinase activities. Following ligand activation, the receptor kinase phosphorylates Smads family, then, they form functional compounds that move into the nucleus to stimulate the transcription of target genes.
Accumulating evidences indicated that BMP-2 has an important role in the development of nervous system (NS), especially in the differentiation, maturation and
apoptosis of neural stem cells. Expressions of BMP-2 and BMPRs in the developing nervous system suggested that BMP-2 could affect proliferation and differentiation of NSCs. At the same time, some evidences indicated that BMP-2 could induce NSCs differentiating into neurons depending on the concentration. Moreover, it was considered that BMP-2 regulates the differentiation of NSCs through Smad signaling pathways (described above). Nevertheless, the effects of BMP-2 on the SVZa NSCs differentiation have not been clearly studied while they migrate from SVZa into OB through RMS. Although there were reports that rhBMP-2 can efficiently induce neuronal differentiation of SVZa NSCs in vitro, the precise mechanism of it is still unknown.
In our study, we first investigated the expressions of BMP-2 mRNA in SVZa-RMS-OB of rat brain at different developing phases by RT-PCR, then observed the temporal and spatial expression patterns of BMP-2 in SVZa-RMS-OB by immunofluorescent staining of BMP-2, nestin, NF200 and GFAP. The results are as follows:
1. NSCs derived from SVZa migrate from SVZa into OB through RMS in developing rat brain; the expressions of nestin are located manly in the SVZa and its surrounding, the RMS and the center of OB, but nestin positive cells are gradually decreased with the development of rat brain.
2. The expressions of NF and GFAP were low level in SVZa-RMS-OB, their expressions were located around of LV and SVZa, OB, there were almost no expressed NF and GFAP in RMS, but the expressions of NF and GFAP were high around OB. The expressions of NF and GFAP have an increasing trend with the development phrase.
3. The expressions of BMP-2: ①in different time points: The expressions of BMP-2 were higher in embryonic day 14 (E14) than that of postnatal day 0 (P0), postnatal day 7 (P7) and adult in the locations of SVZa, RMS and OB, and there were not significant differences between P0 and P7. The expressions of BMP-2 are lowest in adult. ②in different locations: The expressions of BMP-2
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