【作者】 李娜；

【导师】 周莉；

【作者基本信息】 吉林大学 ， 人体解剖与组织胚胎学， 2007， 硕士

【摘要】 目的:研究在端脑发育中骨形态发生蛋白4(BMP-4)对同源域转录因子Nkx2.1表达的影响,探讨Nkx2.1在胆碱能神经元发生过程中的作用以及能否被BMP-4所诱导。方法:将E16大鼠胚胎端脑细胞做体外无血清原代培养,用不同浓度的BMP-4诱导胆碱能神经元的分化,采取免疫细胞化学染色实验观察不同浓度BMP-4对ChAT阳性免疫反应细胞数量的影响。在实验过程中采取对胆碱能神经元分化影响最大的BMP-4浓度,加入到体外培养的E16大鼠胚胎端脑细胞中,进行RT-PCR检测,以研究Nkx2.1在端脑发育中BMP-4对Nkx2.1基因表达的影响。结果:不同浓度BMP-4对胆碱能神经元发生的影响有所不同。低浓度(10ng/ml、20ng/ml)BMP-4可使ChAT阳性细胞数量增多;高浓度(40ng/ml)BMP-4可使ChAT阳性细胞数量减少。RT-PCR结果显示实验组和对照组Nkx2.1基因表达没有明显区别。结论:(1)在体外培养的E16端脑细胞中,低浓度(20ng/ml)BMP-4能促进胆碱能前体细胞的发生和分化;(2)在体外培养的E16端脑细胞中,高浓度(40ng/ml)BMP-4能抑制胆碱能前体细胞的发生和分化;(3)BMP-4细胞外信号分子不能调节E16端脑细胞Nkx2.1 mRNA的表达。更多还原

【Abstract】 The research on generation and development of cholinergic neurons is the base of how to recognize and understand the function of central nerve system. And it is also the hot problem of the research in the field of nerve science. Recently , we have already known much of the development of telencephalon’s generation not only in the level of cell but also in the level of molecule. But we still don’t know clearly the mechanism of generation and development in developing telencephalon.The dorsal telencephalon is the anlage of the developing pallium. The source of cells in different places of telencephalon is generation area,consisted of ventricular zone and subventricular zone. Radial glia in ventricle zone (VZ) is 98% in neurogenesis period,they are the progenitor cells of cholinergic neurons. Choline acetyltransferase(ChAT)is a marker for cholinergic neurons and it can show the generation、transplantation and distribution of cholinergic neurons.We take advantage of Vimentin of radial glia and ChAT to do the experiment of immunohistochemistry double pigmentation in the slice of mouse telencephalon.The results hint us that cholinergic neurons in dorsal and ventral telencephalon are coming from radial glia. Radial glia have already gained cholinergic character before it differentiated nerve cells. How do they gain the character? This problem is the core problem of the research on mechanism of cholinergic neurons generation.More and more research productions tell us that there are two ways of how todecide the fate of nerve progenitor cells. One is extracellular information factor evocate effect;the other is hemeodomain transcription factor distribute in telencephalon by inherition. The interaction between yhe two ways decide the differentiation of nerve cells.From the data collected,the extracellular information factor who relate to the development of cholinergic neurons:BMPs,bFGF,NGF,Shh and Wnt ect. The hemeodomain transcription factor who relate to the development of cholinergic neurons:IsL1,Nkx2.1,Lhx8,Vax1 ect.The function of BMPs is to promote the differentiation and limit of cell family character. But the function of Shh is to make cells gain ventral character. In fact,the differentiation of nerve cells in ventral telencephalon is the result of interaction between Shh and BMPs. But BMPs are inhibited by Noggin and folliatatin. The function of BMPs in the decision of cells fate is inducement in developing telencephalon. bFGF can stimulate the multiplication of nerve stem cells in limited area. bFGF is needed in the course of neural former cells change to neurons. The main area in brain of NGF effect is cholinergic system. NGF can promote the generation of cholinergic neurons, strengthen active of ChAT and AchE. The function of Wnt how to control differentiation of nerve former cells is in the lower reaches of a river of BMPs. The induce function of BMPs is not only active related homeodomain transcription factors but also active transfer way of Wnt. This can make the balance of control network.From the data collected, it is supposed that ISL-1 is probably the main homeodomain transcription factor which can control the development of cholinergic neurons. From the result of the experiment,the expression ofISL-1 mRNA is changed by different concentration BMP-4. Low concentration BMP-4 can promote the expression of ISL-1 mRNA. High concentration BMP-4 can inhibit the expression of ISL-1 mRNA. We find that BMP-4 can promote cells to grow in characteristic round clusters and induce ISL-1 mRNA expression. In our investigation,ISL-1 is proved to be one of the homeodomain transcription factors to regulate cholinergic neurons development cooperated with BMP4. The location of Nkx2.1 combine with special DNA sequence is in the startup factor of ChAT genes. Is the expression of Nkx2.1 in the development of cholinergic neurons? The problems are exactly solved by my experiment. Lhx8 is mainly expressed in ventral telencephalon. But the problem of whether its expression by BMP-4 still need to approve. The effect of Vax1 on cholinergic neurons’generation may be control by other homeodomain transcription factors.In order to testify whether the expression of Nkx2.1 mRNA induce by BMP-4,we must make sure useful concentration of BMP-4. We do the experiment of ChAT immunohistochemistry pigmentation and count for positive cells. We observe the effect of ChAT positive cells variety by different concentration BMP-4, then we confirm the concentration of BMP-4 for the next experiment. From experiment’s results,we find that the largest effect on cholinergic neurons variety is under the function of 20ng/ml BMP-4. So we select 20ng/ml BMP-4 to induce the expression of Nkx2.1.From RT-PCR results, we find that BMP4 can not control the expression of Nkx2.1 in telencephalon. It may be caused for Nkx2.1locating in the upriver of BMP4. We can infer,(1) in every developing organ,the control mechanism of the same kind of cellular information factors may be the same. (2) Nkx2.1 can positive control the expression of Lhx6 and Lhx8. Therefore,Nkx2.1 may be indirectly effect the generation of cholinergic neurons by the control of other homeodomain transcription factors.On the other hand, BMP-4 can’t control the expression of Nkx2.1 in developing telencephalon , and it indirectly confirmed that the expression of ISL-1 mRNA may be control the generation and differentiation of cholinergic progenitor cells. So this result gives us a direction to research clearly the molecule mechanism of cholinergic neurons.更多还原