A Study Of The Effect Of Neurogenesis And Neuron Apoptosis Of RhG-CSF After Cerebral Ischemia In Diabetic Rats

ObjectiveDiabetes cerebral infarction is the main reason for human's death and disability. Up to now,there is no effective way to recover the nerves function.Stem cell therapy is the most hopeful method to solve the problem of nerve regeneration.Transplanting MSC to treat cerebral ischemia has got obvious results in animal experiments,but it is too complicated,increase the possibility of infection and it may miss the treatment time. rhG-CSF is a cell factor which is effective in controlling the proliferation, differentiation and survival of bone marrow stem cell.It can mobilize HSC and MSC into blood circulation,and it is effective in treating experimental myocardium ischemia and cerebral ischemia.This thesis is going to discuss the effect and mechanism of rhG-CSF on diabetes cerebral infarction nerve protection,to observe the change of nerve function and brain tissue ultrastructural organization of focal cerebral ischemia diabetes rat after rhG-CSF intervention,to observe the change of G-CSFR,STAT3,pSTAT3,VEGF,IGF-1,BrdU expression of ischemic area,the TUNEL positive apoptosis cell nerve and Bcl-2,VEGF proteinum and gene change,and the condition of cell coexpression BrdU⁺NeuN⁺,BrdU⁺GFAP⁺,to verify whether rhG-CSF can improve the nerve function of diabetes cerebral infarction rat,whether its function is realized by increasing VEGF,IGF-1 expression,the rivalry of cell nerve apoptosis and promoting nerve regeneration,and the ligand-acceptor mutual effect and the possible route of signal transduction.Methods1.Experimental animal model preparation and grouping.Wister rat diabetes model was prepared by 2 dose regimen.After 6 week,rat MCAO model was prepared by line embolism.The model was divided into rhG-CSF intervention group and contrast group at random,and every group was divided into IDEM effect 7d,14d and 21d time point group at random.There are 12 rats at every time point.2.RhG-CSF intervention and BrdU mark.rhG-CSF intervention group rats were given rhG-CSF50μg/kg every day after operation by hypodermic injection for 7,14, and 21 days for each time point group respectively.The contrast group rats were injected the tales doses normal saline.Each group rats were given BrdU10mg/kg.d by peritoneal injection the first day after operation for 7,14,and 21 days respectively to mark the cells in dividing phase.3.Nerve function score.Each rat's neurologic impairment was scored by NSS the first day and the day before they were killed to observe the effect of rhG-CSF on diabetes cerebral ischemia rat's nerve function.4.Observe the brain tissue ultrastructural change by TEM.5.Detect the effect of rhG-CSF on brain tissue nerve cell apoptosis by Tunel staining.6.Detect the effect of rhG-CSF on brain tissue BrdU,G-CSFR,VEGF,IGF-1 expression by immunohistochemistry.7.Detect the effect of rhG-CSF on brain tissue BrdU⁺NeuN⁺,BrdU⁺GFAP⁺ coexpression by IMF.8.Detect the effect of rhG-CSF on brain tissue STAT3,PSTAT3,VEGF,BCL-2 proteinum expression by Western Blot method. 9.Detect the effect of rhG-CSF on brain tissue nerve cell VEGF,BCL-2 gene mRNA expression by RT-PCR.Results1.There is no distinct difference in rhG-CSF intervention group and contrast group in neurologic impairment the first after operation.The score of rhG-CSF intervention group neurologic impairment was much lower than the contrast group(P＜0.01).2.Cell nucleus rupture of membrane,cell nucleus structure abolition,cell nucleus pycnosis,caryotin side collection,membranolysis and cell organ damage were observed under TEM.3.TUNEL staining result showed that rhG-CSF intervention group cerebral ischemia area immune positive cells were much less than the contrast group the 7^th,14^th and 21^st day(P＜0.01).4.Immunohistochemistry staining result showed that rhG-CSF intervention group cerebral ischemia area VEGF immune positive cells optical density value was much more than that of contrast group the 7_th,14^th and 21^st day(P＜0.01);rhG-CSF intervention group cerebral ischemia area IGF-1 immune positive cells optical density value was much more than that of contrast group the 7^th,14^th and 21^st day(P＜0.01); rhG-CSF intervention group cerebral ischemia area BrdU immune positive cells were much more than those of contrast group the 7^th,14^th and 21^st day(P＜0.01);rhG-CSF intervention group cerebral ischemia area G-CSFR immune positive cells optical density value was much more than that of contrast group the 7^th,14^th and 21^st day (P＜0.01).5.IMF double staining result showed that coexpressiom BrdU⁺NeuN⁺ cell can be observed in cerebral ischemia area in rhG-CSF intervention group;Coexpressiom BrdU⁺NeuN⁺ can not be observed in the contrast group.Coexpressiom BrdU⁺GFAP⁺ can not be observed in the rhG-CSF intervention group and contrast group.6.Western blot result showed that there was no distinct difference in brain tissue STAT3 proteinum expression between rhG-CSF intervention group and the contrast group the 7^th,14^th and 21^st day(P＞0.05);rhG-CSF intervention group brain tissue pSTAT3 proteinum expression was much higher than that of the contrast group the 7^th, 14^th and 21^st day(P＜0.01);rhG-CSF intervention group brain tissue Bcl-2 proteinum expression was much higher than that of the contrast group the 7^th,14^th and 21^st day(P＜0.01);rhG-CSF intervention group brain tissue VEGF proteinum expression was much higher than that of the contrast group the 7^th,14^th and 21^st day(P＜0.01);7.RT-PCR result showed that rhG-CSF intervention group brain tissue Bcl-2mRNA expression was much higher than that of the contrast group the 7^th,14^th and 21^st day(P＜0.01);rhG-CSF intervention group brain tissue VEGFmRNA expression was much higher than that of the contrast group the 7^th,14^th and 21^st day(P＜0.01);Conclusion1.RhG-CSF can significantly improve the nerve function of focal cerebral ischemia diabetes rat.2.RhG-CSF can significantly ease the damage of ultrastructural organization of diabetes rat after focal cerebral ischemia.3.RhG-CSF can significantly decrease the nerve cells apoptosis of diabetes rat after focal cerebral ischemia and it can obviously increase the mRNA of anti-apoptosis factor Bcl-2 and proteinum expression.4.RhG-CSF can increase VEGF,IGF-1,BrdU,G-CSFR proteinum immunohistochemistry expression of cerebral ischemia area.5.RhG-CSF has no evident effect on STAT3 proteinum expression and it can obviously increase pSTAT3 proteinum expression.6.RhG-CSF can significantly increase the mRNAp of VEGF and proteinum expression.7.RhG-CSF can significantly increase nerve regeneration of diabetes rat after focal cerebral ischemia and stimulate new cells to differentiate to neurone instead of horizontal cell.