| Alzheimer's disease is a kind of neural degenerative disease. Central nerve cholinergic system damage doctrine is more received in its pathogenesis now. Our research group previously found that bone morphogenetic proteins-4 (BMP4) can induce neural stem cells (NSCs) to be cholinergic neuron and up-regulate cholinergic-expression of nerve cells of adult rats' dentate band. But can it only induce neural stem cells (NSCs) to be cholinergic neuron? Can it up-regulate cholinergic-expression of neurons? If BMP4 is injected into dentate band, can it amend spatial memory impairment symptom of AD model rats. This is not be reported in the world.Objective: According to observing effect of BMP4 on NSCs' differentiation and neurons' cholinergic-expression, understand further BMP4' effect of cholinergic induction and differentiation for NSCs and identify whether BMP4 has effect of up-regulation of cholinergic-expression for neurons. According to injecting BMP4 into dentate band, understand further BMP4' effect of up-regulation of cholinergic-expression for neural cells of dentate band of adult rats. According to injecting BMP4 into dentate band, identify whether BMP4 has therapeutical effect on spatial memory impairment of AD model rats.Method: (1) Isolated and cultivated NSCs from dentate band of juvenile rats, and the cultivated cells was demonstrated by immunocytochemistry against Nestin. Then the cells were induced by BMP4 and the expression of choline acetyltransferase (ChAT) of the induced cells was observed by immunocytochemical stain and FITC-labeled cholinergic nerve cells Flow Cytometry. (2) Isolated and cultivated neurons from hippocampus from conceptus age of 15-20 days SD rat's brain, and the cultivated cells were demonstrated by immunocytochemistry against MAP2. Then the neurons were up-regulated by BMP4 and the expression of choline acetyltransferase (ChAT) of the cultivated cells was observed by Immunocytochemical stain and FITC-labeled cholinergic nerve cells Flow Cytometry. (3) BMP4 was injected into adult rat's dentate band. After 28 days, cholinergic expression of nerve cells of dentate band was observed by immunohistochemistry against ChAT. The area of ChAT-positive neurons was measured by Image-prog-plus image analytical system andundertake t test by STATA7.0 software. (4) Set up AD model rats and injected BMP4 into its dentate band. After 28 days, had Mirros water maze experiment for the rats and observed BMP4' effect on spatial memory impairment of AD model rats. Then, brain tissue of dentate band of AD model rats was obtained and made paraffin section for immunohistochemistry against ChAT. The area of ChAT-positive neurons was measured by Image-prog-plus image analytical system and undertake t test by STATA7.0 software.Results: (1) Cultivated NSCs accorded with the morphological character and the proliferative trait for NSCs and the cells had the immuno-marker of NSCs; BMP4 group had the prominent hyper-ratio of ChAT-positive cells than non-BMP4 control group (26.64±1. 47%; 13.42±1.33%, P<0.01). Flow Cytometry showed that the ChAT-positive cells percent of BMP4 group were higher than non-BMP4 control group (23.42%; 7.79%). (2) Cultured neurons accorded with the morphological character and for neurons and the cells have the immuno-marker of neurons; BMP4 group had the prominent hyper-ratio of ChAT-positive cells than non-BMP4 control group(13.3±1.37%; 6.44±0.81%, P<0.01). Flow Cytometry showed that the ChAT-positive cells percent of BMP4 group were higher than non-BMP4 control group(11.48%; 4.92%). (3) There are remarkably differentiations of the area of ChAT-positive neurons of dentate band between injected drugs side and opposite side in BMP4 group (194.78t22.33/zm2; 158.03±36.22/zm2, P<0.01) . There are no differentiations of the area of ChAT-positive neurons of dentate band between two sides in NS control group (142.78±26.58/zm2; 143.76±18.76/zm2 , P>0.05) . The area of ChAT-positive neurons of dentate band of injected drugs side dentate band is prominent more than the same side of NS control groups (194.78±22.33/zm2; 142.78t26.58/zm2, P<0.01) . (4) In Mirros water maze experiment, movement time and swimming path of searching for flat roof for AD model group is the longer than normal group, there are remarkably differentiations[(37.21±19.49s> 903.09±707.04; 13.95±8. 38s^ 290.46±156.60, P<0.01) ]; In immunohistochemistry against ChAT for paraffin section of rats' brain, in AD model group the area of ChAT-positive neurons within dentate band of cutting side was much less than normal groups (68.62t24.73/zm2; 144.65+24.27/zm2, P<0.01) .In Mirros... |
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