摘要
研究端脑发育胆碱能神经元发生的机制是理解中枢神经系统功能,寻找中枢神经系统疾病治疗新靶点的基础,也是探索干细胞定向分化、细胞移植治疗和组织工程修复损伤不可缺少的步骤。本实验从研究胆碱能神经元的胚胎来源入手,逐步深入探索其获得胆碱能表型特征的细胞和分子机制。首先,使用流式细胞术确定了胚胎大鼠端脑胆碱能神经元发生的时间;采用形态学和体外细胞培养技术证实了大脑皮层和基底神经节胆碱能神经元分别来源于端脑背侧和腹侧放射状胶质细胞群;实验结果还证实第三脑室室管膜细胞产生胆碱能前体细胞,可能形成基底前脑神经核团的Ch5,Ch6胆碱能神经细胞群。其次,通过骨形态发生蛋白-4对胆碱能神经元的诱导作用,证实了在端脑细胞培养体系中不同浓度BMP4对胆碱能神经元发生数量有不同影响,并且利用这一特点,通过改变培养体系中骨形态发生蛋白-4浓度,采用RT-PCR技术找出mRAN表达随之发生变化的同源域转录因子Isl-1。实验结果提示,胆碱能神经元发生是在一定浓度骨形态发生蛋白-4诱导下神经祖细胞表达LIM同源域转录因子Isl-1,并与胆碱能基因座启动子的特定位点结合,启动胆碱能基因座转录的结果。
Embryonic and adult neural stem cells have been being hot problemsof neuroscience research. Because neural progenitor cells intelencephalon characterize specialization of regional identitydifferentiated potency of the neural stem cells has been restricted by theirdistribution in space-time of developing telencephalon, thereforeapplication of neural stem cell also has been limited in vitro. Howerve,the increasing evidences showed that when neural stem cells weretransplanted into the recipient they have great plasticity. Stem cellstherapy strategy base on induce stem cells orientation differentiate.Investigation of biology property and differentiate mechanism of stemcell are required in normal embryonic development. It will be possiblethat guide application of stem cells if its regularity is understanded, whichis also the basis of understanding the function of center nerve system andsearches for a pathway of the therapy methods of degenerate deseases. Toachieve the aim, firstly research on location of cholinergic neuron inspace-time, origin of cholinergic neuron, and how to obtain cholinergicphenotype during embryonic development are required. This experimenthas been divided into three parts, the first one is investigation of origin ofcholinergic neuron in developing telencephalon, the second one isresearch induce activity of different concentration of BMP4 forgeneration of cholinergic neuron, and the third one is whether expressionof the LIM homeodomain transcription factor Isl-1 are regulated by the BMP4 for generation of cholinergic neuron.
Firstly, this experiment began with demonstrating time of emerging cholinergic neuron and its origin in developing rat telencephalo, telencephalon cells suspension of E10, E11 and E12 rats respectively were prepared, and the cells labeled by anti-choline acetyltransferase antibody (ChAT is a rate-limiting enzyme of synthesizing neurotransmitter acetylcholine, as a protein marker of cholinergic neuron) and FITC-IgG measured by using the flow cytometer. The result showed that the amount of cells fluorescent-labelled for the E12 rat were 22.49%, without statistics significance for the E10 and E11 rat as compared with the negative control, indicating that generation of cholinergic neuron of telencephalon begin in E12.
In 1989 U.B.Schambra and his colleague discovered in their experiments that dividing cells expressed ChAT emerger in germinal zone along ventricle zone (VZ) in developing telencephalon. Nevertheless, traditional view is that neuroepithelial cells differentiate into neuroblast after mitosis of them in VZ, and start to migrate until they reach at destination, at the same time obtain cholinergic phenotype. Once obtained cholinergic phenotype cells no longer have mitosis for developing telencephalon. But SC. Noctor and his research group discovered that radial glial has not only of glial cells properties but also neural stem cells properties. Pyramid neurons of cerebrum cortex were generated by radial glial cells in VZ of dorsal telencephalon, this conclusion breaken previous view. We presume that the expressed ChAT and dividing cells in VZ observed by U.B.Schambra is radial glial cells, the cells may generate cholinergic neuron. To confirm the presume this experiment bases on observing morphology and ultrastructure of radial glial cells, and Whether vimentin (a protein marker of radial glial cells) and ChAT ( a protein marker of cholinergic neuron) located at same cells to determine that radial glial cells generate cholinergic neuron in developing telencephalon., which was examined by using immunoflucesence histochemistry double stainning. The result showed that there are the population of vimentin-positive cells contained ChAT-immunoreactivity in anterior angle VZ of dorsal telencephalon, also, the other ones located at lateral ganglionic eminence of ventral telencephalon, suggest that cholinergic neurons of cerebral cortex are derived from the population of radial glial cells in VZ anterior angle of dorsal telencephalon, and cholinergic neurons of striatum were generated from lateral ganglionic eminence of ventral telencephalon.
The increasing evidences revealed that it has two aspect of factors that determining differentiation of neural progenitor cells, the first one is induction of extracellular informational molecules, the second one is regional expression of homeodomain transcription factors in developing telencephalon, which originates from heredity, the consequence which extracellular informational molecules and homeodomain transcription factors interact ditermines that neural progenitor cells fate. In 2000 Ignacio Lopez-Coviella reported that low concentration of BMP9 can upregulate expression of ChAT by directly acting on ChAT immunoreactive cells of E14 mouse telencephalon in vivo and vitro. The results suggest that BMPs is a key inductive factor and play a important role for generation of cholinergic neuron in telencephalon. BMPs have complex contribution, and the distribution and the local concentration of BMPs in telencephalon vary according to gestational age. To get the information which different concentrations of BMPs affect generation of cholinergic neuron in cultured telencepgalonic cells and quantification analysis of ChAT-positive by using immunocytochemistry staining was performed. The result indicates that the final concentration of 20ng/ml BMP4 does not only increase the quantity of ChAT-positive cells as compared with the control, but also ChAT-positive cells with neurits were observed, in contrast, 40ng/ml BMP4 decreases the quantity of ChAT-positive cells, the ones with neurits were not observed, 10ng/ml BMP4 does not affect quantity of ChAT-positive cells as compared with the control. Some of other evidences elaborate that the higher concentration of BMP inhibits proliferation of cultured telencephalonic cells and leads to apoptosis of neural progenitor cells. This may be why 40ng/ml BMP4 reduces the quantity of cholinergic progenitor cells. It is reported that the effects of BMP4 on generation of cholinergic progenitor cells in vivo was regulated by its inhibitors such as noggin, chordin and follistatint. Extracellular informational molecules Shh participates in the process in ventral telencephalon, which make telencephalonic neural progenitor cells obtain ventral identity. Activity of Shh has a reducing gradient from dorsal to ventral telencephalon, because BMPs signal remarkably alters the response of neural progenitor cells to Shh, and BMPs inhibitor follistatin can elicit dorsal-ventral switch for neural progenitor cells fate. As it seen from the evidences, integration of BMPs and Shh signals determines neural progenitor cells fate of ventral telencephalon in vivo, so BMP play a crucial role in the program. The different concentrations of BMP4 have different influence during peak time of neuron development is fund in this experiment, or 20ng/ml BMP4 increases the quantity of cholinergic progenitor cells and promotes their differentiation. The evidences provide a cue for search of homeodomain transcription factors triggered generation of cholinergic neuron in developing telencephalon.
It is crucial procedure that homeodomain transcription factors triggered generation of cholinergic neuron. Six homeodomain transcription factors related cholinergic neuron are expressed in medial ganglionic eminence of ventral telencephalon, or Nkx2.1, Lhx8/6, Gbx1/2 and Isl-1. We know from internet web of bioinformation that specific sequence of binding site of DNA exists on the promoter zone of cholinergic gene locus for Nkx2-1, Vax1, Isl-1 and Lhx8 homeodemain helix-loop-helix (bHLH) proteins, then using same methord searching the information which cholinergic gene locus may combine the homeodomain transcription factors. The result showed that only Isl-1 exists a binding site on 108 base sequence of cholinergic gene locus (TCCTTAA) among the four homeodomain HLH proteins, also Smad4 does a binding site on 112 base sequence of cholinergic gene locus (TAATTTTTAAT) . Therefore it is possible that LIM homeodomain proteins Isl-1 and product of BMPs signaling pathway Smad4 directly combine with the promoter zone of cholinergic gene locus of neural progenitor cells.
As above data mentioned, we think that Isl-1 may be a key homeodomain transcripton factor triggered generation of cholinergic neuron. In this experiment it is utilized that different concentrations of BMP4 have different influence during peak time of neuron development, to comfirm whether expression of Isl-lmRNA vary with concentration of BMP4 so that to understand the relationship of expression of Isl-1mRNA and generation of cholinergic neuron. It was showed that 20ng/ml BMP4 remarkablely upregulate expression of Isl-1mRNA as compared with the control without change for 10ng/ml BMP4, 40ng/ml BMP4 significantly downregulate expression of Isl-1mRNA, indicating a causality between the effects of BMP4 signaling molecule on generation of cholinergic neuron and expression of Isl-1mRNA, suggest that Isl-1 is a main homeodemain transcription factor for control generation of cholinergic neuron.
But it was reported that Isl-1 is only expressed in cholinergic interneuron of ventral telencephalon, it is not unique homeodomain transcription factor triggered generation of cholinergic neuron, maybe combine with other LIM homeodomain protein and form LIM code to start generation of cholinergic neuron. Therefore, author of this paper supposed that some concentration of BMP4 in local area of ventral telencepgalon combines with BMP receptors on membrane of neural progenitor cells during neuron development period, expression of Isl-1 is upregulated through BMP signal conduction pathway, then the Isl-1 and Lhx8 form LIM code and cholinergic gene start to transcript so that neural progenitoe cells differentiate into cholinergic interneuron of ventral telencephalon.
引文
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