| It has been proved by many researches that neural stem cells (NSCs) has capacity of self-renewing and multi-potential of differentiation. Different kinds of cytokines give its effect during the course of NSCs' proliferation and differentiation. Neurons differentiated from NSCs can express many neurotransmitters, such as GABA, substance P, dopamine, et al. Whereas the vast majority NSCs natural differentiated into GABA-nergic neurons without being given special induction, Seldom does NSCs natural differentiated into other kinds of neurons. Many researches showed that NSCs could change its differentiate directions and differentiate into some special kind of cells when being given some cytokines. It has been reported that bone morphogenetic proteins-9 (BMP9) could induce the expression of the choline acetyltransferase and the vesicular acetylcholine transporter gene, so to up-regulate the acetylcholine synthesis of neurons; intracerebroventricular injection of BMP9 increased acetylcholine levels in vivo. These indicate that BMP9 is a cholinergic differentiating factor for central nervous system. Whether BMP4 - a member of BMPs family, could induce the cholinergic-differentiation for NSCs and up-regulate the cholinergic-expression for neurons haven't been reported by now. In this study we tried to know whether BMP4 has that effect by cells culture in vitro and animal experiment in vivo.At first, NSCs was isolated from hippocampus, striatum, VZ and SVZ from 2 months' SD rat's brain. Cells dispersed with the method ofmechanical separation and chemical digestion was cultured with serum-free medium, and was demonstrated by immunocytochemistry against Nestin, GFAP and NF-160. The results showed that the cultured cells accord with the morphological character and the proliferate trait for NSCs; and all round cells was Nestin-positive, GFAP-negative and NF-160-negative, it means that the cells has the immuno-character of NSCs.Then, adding BMP4 into medium or not, observe the differentiation of NSCs. NSCs was assigned to two groups, BMP4 group was changed to be cultivated with a BMP4-added medium, non-BMP4 group was cultivated with the primary medium as a control group. After 8 days, the expression of choline acetyltransferase (ChAT) of the cultivated cells was observed by indirect immunofluorescence test , double-labeling immunocytochemistry test against ChAT and Nestin and FITC-labeled Flow Cytometry. Results showed that more positive cells with stronger fluorescence intensity been found in the BMP4 group; while less positive cells with weaker fluorescence intensity been found in the non-BMP4 group. There are three kinds of cells in the double-labeling immunocytochemistry test: one kind is ChAT-positive cells, its cytoplasm is indigo, and some cells thereinto appears typical morphological character of neuron; the second kind is Nestin-positive cells, its cytoplasm is red, it seems round or progenitor cells-like, and some of them is dividing; the third kind is neither ChAT-positive nor Nestin-positive cells, with neuron-like character and unstained cytoplasm.Counted under microscope, the cell percent of BMP4 group is: ChAT-positive cells, 28%, Nestin-positive cells, 38%; while the percent of non-BMP4 group is: ChAT-positive cells, 8%; Nestin-positive cells, 52%. The results of Flow Cytometry showed that the BMP4 group have the prominent hyper-ratio of ChAT-positive cells than non-BMP4 group (16%, 7%, P<0.05). From the results, we can see that BMP4 have the function of Cholinergic-inducing for NSCs isolated from rat's hippocampus, striatum and cultured in vitro.Third, BMP4 was injected into adult rat's dentate gyrus to observe its effect for cholinergic-expression of neuron. Twenty adult male SD rats were random assigned to two groups: BMP4 group and control group. Rats were deeply anesthetized, and then being stereotaxically injected 1 Ml BMP4 (BMP4 group) or saline(control group) into their right dentate gyrus. After 14 days, their brains were made paraffin section, then processed for immunohistochernistry again... |
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