中樞神經系統(tǒng)損傷后細胞增殖效應的實驗研究
發(fā)布時間：2018-07-15 10:34
【摘要】： 成年動物中樞神經系統(tǒng)(Central Nervous Systerm,CNS)損傷后形成的膠質瘢痕是CNS再生失敗的重要因素。既往的觀點認為,成熟的膠質瘢痕是由細胞外成分(如IV型膠原和層粘連蛋白)和細胞成分(星形膠質細胞、成纖維細胞、巨噬細胞)等共同構成,其中反應性增生的星形膠質細胞是主要細胞成分。少突膠質前體細胞(oligodendrocyte precursor cell,OPC)是最近發(fā)現(xiàn)的CNS中除神經元、星形膠質細胞、少突膠質細胞、小膠質細胞之外的第五類細胞[1]。然而目前關于OPC是否參于CNS對損傷的反應及其意義方面的研究還較少。20世紀90年代初提出成年哺乳動物CNS存在神經干細胞(Neural Stem Cell, NSC)[2],并且大量的研究證實,NSC存在于CNS的廣泛區(qū)域,這些NSC在體內大多處于靜止狀態(tài),在CNS損傷等病理條件或外界信號分子的刺激下可以被激活并增殖。側腦室壁的腦室下層(subventricular zone,SVZ)和海馬齒狀回的顆粒下層(subgranular zone,SGZ),是成體哺乳動物CNS中兩個NSC富集區(qū)域。Parent等的研究證明多種腦損傷模型,可引起損傷遠隔部位海馬齒狀回和室下區(qū)NSC的增殖,并且增殖的細胞可以分化為星形膠質細胞和神經元等[3]’[4]’[5]’[6]。故CNS損傷不僅可引起損傷部位膠質細胞的活化,還可以導致?lián)p傷部位及遠隔部位NSCs的增殖。近年越來越多的研究顯示,成年哺乳動物NSC只有在特定信號分子存在的微環(huán)境中才能分化為神經元[7]。有研究顯示,BMP4與Noggin是一對對NSC的增殖分化有重要調控作用的蛋白分子。Lim等證實BMP4與Noggin共同調控成年室下區(qū)神經干細胞的分化,BMPs信號通過促進室下區(qū)NSC向星形膠質細胞分化,而抑制神經元的產生[8]。我們的研究發(fā)現(xiàn)在成年動物海馬部位BMP4亦具有促進NSC向星形膠質細胞方向分化的功能[9]。然而BMP4與Noggin在除海馬和室下區(qū)以外的其他部位是否具有類似的作用,BMP4與Noggin對CNS損傷后增殖細胞的分化有何作用,還有待進一步的研究證實。 本研究采用大鼠單側眼球摘除作為動物模型,觀察損傷后不同時間點對側上丘GFAP、NG2陽性細胞的表達變化,證實單側眼球摘除后確實存在腦內其投射部位對側上丘的損傷并闡明GFAP陽性的星形膠質細胞、NG2陽性的OPC增殖的意義。第二部分采用BrdU標記的方法,觀察單側眼球摘除后損傷部位、海馬齒狀回和室下區(qū)BrdU陽性細胞的增殖,結合BrdU/GFAP、BrdU/DCX免疫熒光雙標,探討增殖陽性細胞的分化方向。最后一部分為進一步探討影響CNS損傷后損傷部位增殖細胞分化的因素,采用原位雜交和Western-blot檢測損傷后不同時間點對側上丘B(yǎng)MP4 mRNA與Noggin mRNA的表達變化及Noggin蛋白的表達變化。 主要結果: 1.單側眼球摘除后24h對側上丘GFAP染色較正常對照組增加,術后24h時到1w這種表現(xiàn)進行性增加,至1w時上丘的GFAP陽性細胞數量達到頂峰,染色加深,突起增多增長。但術后1w到3w之間,GFAP染色并不進一步增加。 2.單側眼球摘除后24h對側上丘NG2染色加深,損傷后1w NG2陽性細胞數量達高峰,1w至3w之間NG2陽性細胞數量緩慢減少。 3.采用BrdU標記的方法,我們發(fā)現(xiàn)單側眼球摘除后24h視神經的投射部位對側上丘除血管內皮細胞外出現(xiàn)少量BrdU陽性細胞,1w時陽性細胞數量達高峰;損傷后2w時陽性細胞數量較1w時減少,3w時BrdU陽性細胞進一步減少。 4.單側眼球摘除后24h雙側海馬齒狀回和室下區(qū)BrdU陽性細胞數量與對照組相比輕度升高,1w時BrdU陽性細胞數量達到高峰,2w至3w之間BrdU陽性細胞數量逐漸減少。除此之外我們還觀察到,損傷后2w時,側腦室背外側角內陽性細胞數量增加,向外延伸的距離也明顯增加;胼胝體內BrdU陽性細胞數量增多。 5.單側眼球摘除后1w雙側海馬齒狀回和室下區(qū)DCX陽性細胞與對照組比較明顯增多,隨后的兩周陽性細胞數量減少,3w時降至正常對照組水平。 6.免疫熒光雙標顯示:對側上丘增殖的BrdU陽性細胞中可檢測到少量BrdU/GFAP雙標陽性細胞,無BrdU/DCX雙標陽性細胞。海馬齒狀回和室下區(qū)增殖的BrdU陽性細胞存在BrdU/GFAP陽雙標性細胞和BrdU/DCX雙標陽性細胞。 7.BMP4 mRNA原位雜交實驗發(fā)現(xiàn),單側眼球摘除后24h對側上丘B(yǎng)MP4 mRNA陽性細胞數量較正常對照組明顯增升高,染色加深,1w組BMP4mRNA的表達與24h組相比已開始降低,2w3w組BMP4mRNA陽性細胞數量進一步減少,3w時降至正常水平。 8.各組對側上丘Noggin mRNA的表達無明顯差異,1w、2w3w組與正常對照組相比均無明顯變化,Western-blot實驗結果也發(fā)現(xiàn),Noggin蛋白的表達在各組無明顯差異。 主要結論: 1.我們觀察到單側眼球摘除后對側上丘GFAP陽性的星形膠質細胞反應性增生、NG2陽性的OPC活化,證實單側眼球摘除后對側上丘確實存在損傷反應,建立了一個新的、簡便可行的CNS損傷的動物模型;同時證實除星形膠質細胞反應性增生外,CNS對損傷的反應還包括NG2陽性的OPC活化。 2.CNS損傷不僅可以引起損傷局部包括星形膠質細胞和OPC在內的膠質細胞反應,還可以誘導損傷局部出現(xiàn)BrdU陽性細胞,海馬齒狀回和室下區(qū)BrdU陽性細胞數量增加,這些部位增殖的BrdU陽性細胞中存在星形膠質細胞和神經元前體細胞。 3.CNS損傷后BMP4與Noggin的表達可能是調控損傷后損傷部位增殖細胞分化的重要因素。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R363
本文編號：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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R363
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