【作者】 兰希发；

【导师】 张朝东；

【作者基本信息】 中国医科大学 ， 神经病学， 2008， 博士

【摘要】 目的糖尿病合并脑梗死是引起人类死亡和残疾的主要原因,目前尚缺少促进神经功能恢复的有效方法。干细胞疗法是最有希望解决神经再生的方法。骨髓基质细胞移植治疗脑缺血在动物实验研究中已经取得了明显的效果,但它仍然是一种复杂的操作,增加了感染的机会,并且有可能错过治疗的时间窗。重组人粒细胞集落刺激因子(rhG-CSF)是一种对调控骨髓干细胞增生、分化和存活有显著作用的细胞因子,可动员造血干细胞和骨髓基质细胞进入血循环,在治疗实验性心肌缺血和脑缺血方面效果显著。本实验试图从rhG-CSF动员骨髓干细胞,激活脑内神经干细胞的角度探讨rhG-CSF对糖尿病合并脑梗死的神经保护作用及其机制,观察经过rhG-CSF干预之后,局灶性脑缺血糖尿病大鼠的神经功能和脑组织超微结构的变化,缺血周边区G-CSFR、STAT3、pSTAT3、VEGF、IGF-1、BrdU表达的变化,TUNEL阳性的凋亡神经细胞及Bcl-2、VEGF的蛋白和基因变化,以及细胞共表达BrdU~+NeuN~+、BrdU~+GFAP~+的情况,验证rhG-CSF能否改善糖尿病大鼠合并脑梗死的神经功能,其作用是否通过增加VEGF、IGF-1的表达,拮抗神经细胞凋亡和促进神经再生而实现,以及其配体-受体的相互作用和信号转导的可能途径。方法1、实验动物模型制备及分组wister大鼠采用2次给药法制备大鼠糖尿病模型,6周后采用线栓法制备大鼠大脑中动脉闭塞(MCAO)模型。随机分成rhG-CSF干预组、对照组,每组又随机分为缺血7d、14d和21d时间点组,每一时间点均为12只大鼠。2、rhG-CSF干预及BrdU标记rhG-CSF干预组大鼠术后每日皮下注射rhG-CSF50μg/kg,各时间点组按组分别注射7、14和21天。对照组注射等量的生理盐水。各组均从术后第1天开始,腹腔注射BrdU10mg/kg.d,分别连续注射7、14和21天以标记处于分裂期的细胞。3、神经功能评分每只动物均于术后第1天和处死前采用神经功能缺损评分标准(NSS)进行神经功能缺损评分,观察rhG-CSF对糖尿病脑缺血大鼠神经功能的影响。4、透射电镜观察rhG-CSF对脑组织超微结构的改变。5、Tunel染色方法检测rhG-CSF对脑组织中神经细胞凋亡的影响。6、免疫组化方法检测rhG-CSF对脑组织中BrdU、G-CSFR、VEGF、IGF-1的表达的影响。7、免疫荧光检测rhG-CSF对脑组织中细胞共表达BrdU~+NeuN~+、BrdU~+GFAP~+情况的影响。8、Western Blot方法检测rhG-CSF对脑组织中STAT3、pSTAT3、VEGF、BCL-2蛋白表达的影响。9、RT-PCR方法检测rhG-CSF对脑组织中神经细胞VEGF、BCL-2基因的mRNA表达的影响。结果1、rhG-CSF干预组和对照组术后第1天的神经功能缺损评分无明显差异;rhG-CSF干预组第7、14、21天的神经功能缺损评分明显低于对照组(P＜0.01)。2、透射电子显微镜下观察到对照组脑组织缺血后细胞核膜破裂,染色质边聚,细胞膜破裂,各种细胞器破坏。rhG-CSF组也可观察到上述破坏的神经元,但超微结构的损伤明显减轻。3、TUNEL染色结果显示:rhG-CSF干预组第7、14、21天脑缺血周边区TUNEL免疫阳性细胞数明显少于对照组(P＜0.01)。4、免疫组化染色结果显示:rhG-CSF干预组第7、14、21天脑缺血周边区VEGF免疫阳性细胞光密度值明显多于对照组(P＜0.01);rhG-CSF干预组第7、14、21天脑缺血周边区IGF-1免疫阳性细胞光密度值明显多于对照组(P＜0.01);rhG-CSF干预组第7、14、21天脑缺血周边区BrdU免疫阳性细胞数明显多于对照组(P＜0.01);rhG-CSF干预组第7、14、21天脑缺血周边区G-CSFR免疫阳性细胞光密度值明显多于对照组(P＜0.01)。5、免疫荧光双标染色结果显示:rhG-CSF干预组脑缺血周边区可观察到共表达BrdU~+NeuN~+细胞,对照组未观察到共表达BrdU~+NeuN~+细胞;rhG-CSF干预组和对照组都未观察到共表达BrdU~+GFAP~+细胞。6、Western blot结果显示:rhG-CSF干预组第7、14、21天脑组织STAT3蛋白表达与对照组无显著差异(P＞0.05)。rhG-CSF干预组第7、14、21天脑组织pSTAT3蛋白表达明显高于对照组(P＜0.01)。rhG-CSF干预组第7、14、21天脑组织Bcl-2蛋白表达明显高于对照组(P＜0.01)。rhG-CSF干预组第7、14、21天脑组织VEGF蛋白表达明显高于对照组(P＜0.01)。7、RT-PCR结果显示:rhG-CSF干预组第7、14、21天脑组织Bcl-2mRNA表达明显高于对照组(P＜0.01)。rhG-CSF干预组第7、14、21天脑组织VEGFmRNA表达明显高于对照组(P＜0.01)。结论1、rhG-CSF能明显改善糖尿病大鼠局灶性脑缺血后的神经功能。2、rhG-CSF可以明显减轻糖尿病大鼠局灶性脑缺血后的超微结构损害。3、rhG-CSF可以明显减少糖尿病大鼠局灶性脑缺血后的神经细胞凋亡;并能明显增加抗凋亡因子Bcl-2的mRNA和蛋白的表达。4、rhG-CSF可以增加脑缺血周边区的VEGF、IGF-1、BrdU、G-CSFR蛋白的免疫组化表达。5、rhG-CSF对STAT3蛋白的表达无明显影响,能明显增加pSTAT3蛋白的表达。6、rhG-CSF能明显增加VEGF的mRNA和蛋白的表达。7、rhG-CSF能明显增加糖尿病大鼠局灶性脑缺血后的神经再生,促进新生细胞向神经元方向分化,但不促进向星形胶质细胞的分化。更多还原

【Abstract】 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.更多还原