中枢神经系统损伤后细胞增殖效应的实验研究
发布时间：2018-07-15 10:34
【摘要】： 成年动物中枢神经系统(Central Nervous Systerm,CNS)损伤后形成的胶质瘢痕是CNS再生失败的重要因素。既往的观点认为,成熟的胶质瘢痕是由细胞外成分(如IV型胶原和层粘连蛋白)和细胞成分(星形胶质细胞、成纤维细胞、巨噬细胞)等共同构成,其中反应性增生的星形胶质细胞是主要细胞成分。少突胶质前体细胞(oligodendrocyte precursor cell,OPC)是最近发现的CNS中除神经元、星形胶质细胞、少突胶质细胞、小胶质细胞之外的第五类细胞[1]。然而目前关于OPC是否参于CNS对损伤的反应及其意义方面的研究还较少。20世纪90年代初提出成年哺乳动物CNS存在神经干细胞(Neural Stem Cell, NSC)[2],并且大量的研究证实,NSC存在于CNS的广泛区域,这些NSC在体内大多处于静止状态,在CNS损伤等病理条件或外界信号分子的刺激下可以被激活并增殖。侧脑室壁的脑室下层(subventricular zone,SVZ)和海马齿状回的颗粒下层(subgranular zone,SGZ),是成体哺乳动物CNS中两个NSC富集区域。Parent等的研究证明多种脑损伤模型,可引起损伤远隔部位海马齿状回和室下区NSC的增殖,并且增殖的细胞可以分化为星形胶质细胞和神经元等[3]’[4]’[5]’[6]。故CNS损伤不仅可引起损伤部位胶质细胞的活化,还可以导致损伤部位及远隔部位NSCs的增殖。近年越来越多的研究显示,成年哺乳动物NSC只有在特定信号分子存在的微环境中才能分化为神经元[7]。有研究显示,BMP4与Noggin是一对对NSC的增殖分化有重要调控作用的蛋白分子。Lim等证实BMP4与Noggin共同调控成年室下区神经干细胞的分化,BMPs信号通过促进室下区NSC向星形胶质细胞分化,而抑制神经元的产生[8]。我们的研究发现在成年动物海马部位BMP4亦具有促进NSC向星形胶质细胞方向分化的功能[9]。然而BMP4与Noggin在除海马和室下区以外的其他部位是否具有类似的作用,BMP4与Noggin对CNS损伤后增殖细胞的分化有何作用,还有待进一步的研究证实。 本研究采用大鼠单侧眼球摘除作为动物模型,观察损伤后不同时间点对侧上丘GFAP、NG2阳性细胞的表达变化,证实单侧眼球摘除后确实存在脑内其投射部位对侧上丘的损伤并阐明GFAP阳性的星形胶质细胞、NG2阳性的OPC增殖的意义。第二部分采用BrdU标记的方法,观察单侧眼球摘除后损伤部位、海马齿状回和室下区BrdU阳性细胞的增殖,结合BrdU/GFAP、BrdU/DCX免疫荧光双标,探讨增殖阳性细胞的分化方向。最后一部分为进一步探讨影响CNS损伤后损伤部位增殖细胞分化的因素,采用原位杂交和Western-blot检测损伤后不同时间点对侧上丘BMP4 mRNA与Noggin mRNA的表达变化及Noggin蛋白的表达变化。 主要结果: 1.单侧眼球摘除后24h对侧上丘GFAP染色较正常对照组增加,术后24h时到1w这种表现进行性增加,至1w时上丘的GFAP阳性细胞数量达到顶峰,染色加深,突起增多增长。但术后1w到3w之间,GFAP染色并不进一步增加。 2.单侧眼球摘除后24h对侧上丘NG2染色加深,损伤后1w NG2阳性细胞数量达高峰,1w至3w之间NG2阳性细胞数量缓慢减少。 3.采用BrdU标记的方法,我们发现单侧眼球摘除后24h视神经的投射部位对侧上丘除血管内皮细胞外出现少量BrdU阳性细胞,1w时阳性细胞数量达高峰;损伤后2w时阳性细胞数量较1w时减少,3w时BrdU阳性细胞进一步减少。 4.单侧眼球摘除后24h双侧海马齿状回和室下区BrdU阳性细胞数量与对照组相比轻度升高,1w时BrdU阳性细胞数量达到高峰,2w至3w之间BrdU阳性细胞数量逐渐减少。除此之外我们还观察到,损伤后2w时,侧脑室背外侧角内阳性细胞数量增加,向外延伸的距离也明显增加;胼胝体内BrdU阳性细胞数量增多。 5.单侧眼球摘除后1w双侧海马齿状回和室下区DCX阳性细胞与对照组比较明显增多,随后的两周阳性细胞数量减少,3w时降至正常对照组水平。 6.免疫荧光双标显示:对侧上丘增殖的BrdU阳性细胞中可检测到少量BrdU/GFAP双标阳性细胞,无BrdU/DCX双标阳性细胞。海马齿状回和室下区增殖的BrdU阳性细胞存在BrdU/GFAP阳双标性细胞和BrdU/DCX双标阳性细胞。 7.BMP4 mRNA原位杂交实验发现,单侧眼球摘除后24h对侧上丘BMP4 mRNA阳性细胞数量较正常对照组明显增升高,染色加深,1w组BMP4mRNA的表达与24h组相比已开始降低,2w3w组BMP4mRNA阳性细胞数量进一步减少,3w时降至正常水平。 8.各组对侧上丘Noggin mRNA的表达无明显差异,1w、2w3w组与正常对照组相比均无明显变化,Western-blot实验结果也发现,Noggin蛋白的表达在各组无明显差异。 主要结论: 1.我们观察到单侧眼球摘除后对侧上丘GFAP阳性的星形胶质细胞反应性增生、NG2阳性的OPC活化,证实单侧眼球摘除后对侧上丘确实存在损伤反应,建立了一个新的、简便可行的CNS损伤的动物模型;同时证实除星形胶质细胞反应性增生外,CNS对损伤的反应还包括NG2阳性的OPC活化。 2.CNS损伤不仅可以引起损伤局部包括星形胶质细胞和OPC在内的胶质细胞反应,还可以诱导损伤局部出现BrdU阳性细胞,海马齿状回和室下区BrdU阳性细胞数量增加,这些部位增殖的BrdU阳性细胞中存在星形胶质细胞和神经元前体细胞。 3.CNS损伤后BMP4与Noggin的表达可能是调控损伤后损伤部位增殖细胞分化的重要因素。
[Abstract]:The glial scar formed after the Central Nervous Systerm (CNS) injury in adult animals is an important factor in the failure of CNS regeneration. In the past, the mature glial scar is the co structure of extracellular components (such as collagen type IV and laminin) and cell components (astrocytes, fibroblasts, macrophages) The reactive proliferation of astrocytes is the main cell component. Oligodendrocyte precursor cell (OPC) is a recent discovery of the fifth kinds of fine cell [1]. except neurons, astrocytes, oligodendrocytes, and microglia, but it is currently about whether OPC is involved in CNS against damage. The study of its significance and its significance is still less in the early 90s of the.20 century. It was proposed that adult mammalian CNS exist neural stem cells (Neural Stem Cell, NSC) [2], and a large number of studies have confirmed that NSC exists in a wide range of CNS, and these NSC are mostly in a state of rest in the body, stimulated by pathological conditions such as CNS injury or by external signal molecules. To be activated and proliferate, the lower layer of the ventricle (subventricular zone, SVZ) and the underlayer of the dentate gyrus of the lateral ventricle (subgranular zone, SGZ) are the.Parent in the two NSC enrichment regions of the adult mammalian CNS, which prove a variety of brain damage models, which can cause the proliferation of NSC in the dentate gyrus and subventricular zone of the damaged region. The proliferating cells can differentiate into [3] '[4]' [5] '[6]., such as astrocytes and neurons, so CNS damage can not only cause the activation of glial cells in damaged sites, but also lead to the proliferation of NSCs in damaged sites and distant sites. In recent years, more and more studies have shown that adult mammalian activity NSC is only microring existing in specific signal molecules. BMP4 and Noggin are [7]., a pair of protein molecules that play an important role in the proliferation and differentiation of NSC, which confirm that BMP4 and Noggin regulate the differentiation of neural stem cells in the subventricular region by BMP4 and Noggin, and the BMPs signal differentiates into astrocytes by promoting the NSC to the astrocytes in the subventricular region, and inhibits the production of [8]. Our study found that BMP4 also has the function of promoting the differentiation of NSC into astrocytes in the hippocampus of adult animals, [9]., however, whether BMP4 and Noggin have a similar role in other parts except the hippocampus and subventricular region. What is the effect of BMP4 and Noggin on the differentiation of proliferating cells after CNS damage? There is still to be further study Confirm.
In this study, unilateral exucleation of the rat was used as an animal model to observe the expression of GFAP and NG2 positive cells in the lateral superior colliculus at different time points after the injury. It was confirmed that the unilateral exucleation of the lateral colliculus after unilateral exucleation of the brain existed in the brain, and the GFAP positive astrocytes, and the significance of the proliferation of NG2 positive OPC, were found in the second part. BrdU markers were used to observe the proliferation of BrdU positive cells in the dentate gyrus and subventricular zone after unilateral exucleation of the eyeball, combined with BrdU/GFAP and BrdU/DCX immunofluorescence, to explore the differentiation direction of the proliferation positive cells. The last part was to further explore the factors affecting the differentiation of the proliferating cells after the injury of CNS. In situ hybridization and Western-blot were used to detect the expression of BMP4 mRNA and Noggin mRNA in the superior colliculus at different time points after injury and the expression of Noggin protein.
Main results:
1. after unilateral exucleation, the GFAP staining of the lateral superior colliculus increased in 24h than that in the normal control group. The expression of 24h to 1W increased after the operation, and the number of GFAP positive cells in the upper colliculus reached the peak at 1W, and the dyeing deepened and the prominences increased. But the GFAP staining did not increase step by step between 1W to 3W after the operation.
2. after unilateral enucleation, the NG2 staining of 24h superior colliculus increased. The number of 1W NG2 positive cells reached a peak after injury, and the number of NG2 positive cells decreased slowly from 1W to 3W.
3. using the BrdU labeling method, we found that a small amount of BrdU positive cells were found outside the vascular endothelial cells of the lateral colliculus after unilateral exucleation of the 24h, and the number of positive cells reached the peak at 1W, and the number of positive cells decreased at 2W after 2W, and the BrdU positive cells decreased further at 3W.
4. after unilateral exucleation, the number of BrdU positive cells in the dentate gyrus and subventricular zone of 24h was slightly higher than that in the control group. The number of BrdU positive cells reached the peak at 1W, and the number of BrdU positive cells decreased gradually between 2W and 3W. Besides, we observed that the number of positive cells in the lateral ventricle of the lateral ventricle increased after 2W. The number of BrdU positive cells in corpus callosum increased.
After 5. unilateral exucleation, the DCX positive cells in the dentate gyrus and subventricular zone of 1W were significantly increased, the number of positive cells in the subsequent two weeks decreased and the level of 3W decreased to the normal control group.
6. the double immunofluorescence double labeling showed that a small amount of BrdU/GFAP double standard positive cells were detected in the BrdU positive cells with the proliferation of the superior colliculus, and there was no BrdU/DCX double standard positive cells. The BrdU positive cells of the hippocampal dentate gyrus and subventricular zone had BrdU/GFAP positive double standard cells and BrdU/DCX double standard positive cells.
The 7.BMP4 mRNA in situ hybridization showed that the number of BMP4 mRNA positive cells in the lateral superior colliculus increased significantly after the unilateral exucleation of the 24h, and the staining was deepened. The expression of BMP4mRNA in the 1W group was lower than that of the 24h group. The number of BMP4mRNA positive cells in the 2W? 3W group was further reduced, and the 3W was reduced to the normal level.
8. the expression of Noggin mRNA in the lateral superior colliculus was not significantly different in each group. There was no significant change in 1W, 2W? 3W group compared with the normal control group. The results of Western-blot experiment also found that there was no significant difference in the expression of Noggin protein in each group.
The main conclusions are as follows:
1. we observed the reactive proliferation of GFAP positive astrocytes in the lateral superior colliculus after unilateral exucleation and the activation of NG2 positive OPC. It was confirmed that the unilateral exucleation of the lateral superior colliculus did have a damage response to the lateral superior colliculus, and established a new, simple and feasible animal model for the CNS damage; meanwhile, it was confirmed that the astrocytes were reactive proliferation except the astrocyte reactive hyperplasia, CN The response of S to injury also includes the activation of NG2 positive OPC.
2.CNS damage can not only induce local response to glial cells, including astrocytes and OPC, but also induce local BrdU positive cells. The number of BrdU positive cells in the dentate gyrus and subventricular zone in the hippocampus increases, and the BrdU positive cells proliferate in astrocytes and neuron progenitor cells.
The expression of BMP4 and Noggin after 3.CNS injury may be an important factor in regulating proliferation of differentiated cells after injury.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363
本文编号：2123784
[Abstract]:The glial scar formed after the Central Nervous Systerm (CNS) injury in adult animals is an important factor in the failure of CNS regeneration. In the past, the mature glial scar is the co structure of extracellular components (such as collagen type IV and laminin) and cell components (astrocytes, fibroblasts, macrophages) The reactive proliferation of astrocytes is the main cell component. Oligodendrocyte precursor cell (OPC) is a recent discovery of the fifth kinds of fine cell [1]. except neurons, astrocytes, oligodendrocytes, and microglia, but it is currently about whether OPC is involved in CNS against damage. The study of its significance and its significance is still less in the early 90s of the.20 century. It was proposed that adult mammalian CNS exist neural stem cells (Neural Stem Cell, NSC) [2], and a large number of studies have confirmed that NSC exists in a wide range of CNS, and these NSC are mostly in a state of rest in the body, stimulated by pathological conditions such as CNS injury or by external signal molecules. To be activated and proliferate, the lower layer of the ventricle (subventricular zone, SVZ) and the underlayer of the dentate gyrus of the lateral ventricle (subgranular zone, SGZ) are the.Parent in the two NSC enrichment regions of the adult mammalian CNS, which prove a variety of brain damage models, which can cause the proliferation of NSC in the dentate gyrus and subventricular zone of the damaged region. The proliferating cells can differentiate into [3] '[4]' [5] '[6]., such as astrocytes and neurons, so CNS damage can not only cause the activation of glial cells in damaged sites, but also lead to the proliferation of NSCs in damaged sites and distant sites. In recent years, more and more studies have shown that adult mammalian activity NSC is only microring existing in specific signal molecules. BMP4 and Noggin are [7]., a pair of protein molecules that play an important role in the proliferation and differentiation of NSC, which confirm that BMP4 and Noggin regulate the differentiation of neural stem cells in the subventricular region by BMP4 and Noggin, and the BMPs signal differentiates into astrocytes by promoting the NSC to the astrocytes in the subventricular region, and inhibits the production of [8]. Our study found that BMP4 also has the function of promoting the differentiation of NSC into astrocytes in the hippocampus of adult animals, [9]., however, whether BMP4 and Noggin have a similar role in other parts except the hippocampus and subventricular region. What is the effect of BMP4 and Noggin on the differentiation of proliferating cells after CNS damage? There is still to be further study Confirm.
In this study, unilateral exucleation of the rat was used as an animal model to observe the expression of GFAP and NG2 positive cells in the lateral superior colliculus at different time points after the injury. It was confirmed that the unilateral exucleation of the lateral colliculus after unilateral exucleation of the brain existed in the brain, and the GFAP positive astrocytes, and the significance of the proliferation of NG2 positive OPC, were found in the second part. BrdU markers were used to observe the proliferation of BrdU positive cells in the dentate gyrus and subventricular zone after unilateral exucleation of the eyeball, combined with BrdU/GFAP and BrdU/DCX immunofluorescence, to explore the differentiation direction of the proliferation positive cells. The last part was to further explore the factors affecting the differentiation of the proliferating cells after the injury of CNS. In situ hybridization and Western-blot were used to detect the expression of BMP4 mRNA and Noggin mRNA in the superior colliculus at different time points after injury and the expression of Noggin protein.
Main results:
1. after unilateral exucleation, the GFAP staining of the lateral superior colliculus increased in 24h than that in the normal control group. The expression of 24h to 1W increased after the operation, and the number of GFAP positive cells in the upper colliculus reached the peak at 1W, and the dyeing deepened and the prominences increased. But the GFAP staining did not increase step by step between 1W to 3W after the operation.
2. after unilateral enucleation, the NG2 staining of 24h superior colliculus increased. The number of 1W NG2 positive cells reached a peak after injury, and the number of NG2 positive cells decreased slowly from 1W to 3W.
3. using the BrdU labeling method, we found that a small amount of BrdU positive cells were found outside the vascular endothelial cells of the lateral colliculus after unilateral exucleation of the 24h, and the number of positive cells reached the peak at 1W, and the number of positive cells decreased at 2W after 2W, and the BrdU positive cells decreased further at 3W.
4. after unilateral exucleation, the number of BrdU positive cells in the dentate gyrus and subventricular zone of 24h was slightly higher than that in the control group. The number of BrdU positive cells reached the peak at 1W, and the number of BrdU positive cells decreased gradually between 2W and 3W. Besides, we observed that the number of positive cells in the lateral ventricle of the lateral ventricle increased after 2W. The number of BrdU positive cells in corpus callosum increased.
After 5. unilateral exucleation, the DCX positive cells in the dentate gyrus and subventricular zone of 1W were significantly increased, the number of positive cells in the subsequent two weeks decreased and the level of 3W decreased to the normal control group.
6. the double immunofluorescence double labeling showed that a small amount of BrdU/GFAP double standard positive cells were detected in the BrdU positive cells with the proliferation of the superior colliculus, and there was no BrdU/DCX double standard positive cells. The BrdU positive cells of the hippocampal dentate gyrus and subventricular zone had BrdU/GFAP positive double standard cells and BrdU/DCX double standard positive cells.
The 7.BMP4 mRNA in situ hybridization showed that the number of BMP4 mRNA positive cells in the lateral superior colliculus increased significantly after the unilateral exucleation of the 24h, and the staining was deepened. The expression of BMP4mRNA in the 1W group was lower than that of the 24h group. The number of BMP4mRNA positive cells in the 2W? 3W group was further reduced, and the 3W was reduced to the normal level.
8. the expression of Noggin mRNA in the lateral superior colliculus was not significantly different in each group. There was no significant change in 1W, 2W? 3W group compared with the normal control group. The results of Western-blot experiment also found that there was no significant difference in the expression of Noggin protein in each group.
The main conclusions are as follows:
1. we observed the reactive proliferation of GFAP positive astrocytes in the lateral superior colliculus after unilateral exucleation and the activation of NG2 positive OPC. It was confirmed that the unilateral exucleation of the lateral superior colliculus did have a damage response to the lateral superior colliculus, and established a new, simple and feasible animal model for the CNS damage; meanwhile, it was confirmed that the astrocytes were reactive proliferation except the astrocyte reactive hyperplasia, CN The response of S to injury also includes the activation of NG2 positive OPC.
2.CNS damage can not only induce local response to glial cells, including astrocytes and OPC, but also induce local BrdU positive cells. The number of BrdU positive cells in the dentate gyrus and subventricular zone in the hippocampus increases, and the BrdU positive cells proliferate in astrocytes and neuron progenitor cells.
The expression of BMP4 and Noggin after 3.CNS injury may be an important factor in regulating proliferation of differentiated cells after injury.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363
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