硫酸软骨素酶ABC对大鼠脑损伤（TBI）模型胶质瘢痕影响的实验研究
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摘要
脑损伤(traumatic brain injury, TBI)是公认的多发病及难愈的伤病之一,具有高致残率、高耗费、高死亡率等特点。目前在应用各种治疗方法的幸存者中,有大约75%的病员呈不同程度劳动能力丧失,其中重度致残者约占40%,对家庭、社会均造成沉重负担。如何有效地修复脑损伤,迄今为止尚无满意有效的对策,究其原因主要在于已成熟的中枢神经再生困难。然而,随着医学科学的进步,近年来对中枢神经系统(central nervous system, CNS)神经再生的一系列实验研究成果为脑损伤后神经再生带来了新的希望。目前人们对于脑组织损伤后神经再生的认识包括以下几个方面：1、神经细胞本身不能再生,神经干细胞(neural stem cells, NSCs)、胚胎组织或其它移植的细胞能否向脑神经细胞转化进而修复脑损伤尚研究阶段。2、胶质细胞可以再生,并且在损伤后会大量增生,试图“修复”损伤,但大量的胶质细胞增生形成胶质瘢痕,反成为轴突再生的障碍。3、轴突在脑损伤后的再生能力,虽然受到诸多因素(包括抑制因子存在等)影响,但还是具备再生的能力。许多新的方法如神经移植包括周围神经移植、NSCs移植、嗅鞘细胞移植、基因治疗等均可使成年动物受损伤的脑组织表现出某种程度的功能恢复。
脑损伤后会启动一系列与形成瘢痕组织相关的细胞及分子间的反应,形成以星形胶质细胞为主的瘢痕组织,这种致密的胶质瘢痕包囊脑的损伤区从而把脑损伤区与正常脑组织隔开,使再生的神经轴突不能越过损伤区的胶质瘢痕,造成再生的神经轴突不能与突触后神经元建立功能性链接；同时星形胶质细胞分泌的蛋白多糖即硫酸软骨素蛋白多糖(chondroitin sulfate proteoglycans, CSPGs)在损伤区的堆积也是抑制神经再生的重要原因。所以,胶质瘢痕组织是导致脑组织神经难以再生的主要原因之一。
研究证实,CSPGs有非常强的抑制轴突再生功能,而作为细菌胞外裂解酶的硫酸软骨素酶ABC (chondroitin sulfate enzyme ABC, chABC)则可以通过干预CSPGs对神经修复的抑制作用而减少神经胶质瘢痕,促进神经再生。
本课题研究主要内容是：使用自由落体撞击法建立脑损伤胶质瘢痕的动物模型,并依照分组条件在治疗组的动物损伤点上即时注射不同浓度的chABC;在此后的1周、2周及4周三个时间点取出各组脑组织标本,行镜下比较观察各组(包括正常组、模型组对照组、治疗组)的损伤点星形胶质细胞聚集程度以了解瘢痕组织生长情况,并以免疫组化和蛋白印迹观察CSPGs及胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)的表达情况,以判断去瘢痕的治疗效果。
第一部分大鼠脑损伤后胶质瘢痕模型的制作
目的：构建大鼠脑损伤胶质瘢痕模型,为后续进一步对去瘢痕组织治疗研究奠定基础。
方法：wistar大鼠随机分为4组,每组9只,分别为模型对照组、治疗组1(1U/ml ChABC)、治疗组2(2.5U/ml ChABC)、治疗组3(5U/ml ChABC);另还有2只为正常对照组。采用改进的Feeney法按自由落体原理制造成大鼠重度TBI模型。撞击锤重20 g,下落高度30 cm,落体致伤的冲击力为20×30 g·cm。将大鼠头部固定于立体头架上,沿头皮中线矢状位切开皮肤约2 cm,然后逐层切开皮下筋膜,分离骨膜至颅骨,以前囱尾侧2mm、矢状缝左侧2mm的左侧大脑皮质后肢运动区域为中心,开一直径为5mm的圆形骨窗,硬膜保持完整。20 g撞击捶从30cm高的外周套管下落,撞击已置于骨窗硬膜上的撞杆；撞击伤后,充分止血即缝合头皮。治疗组则即时在损伤点注射不同浓度的chABC。
结果：以Feeney自由落体撞击法建立了重度大鼠TBI模型,为大鼠TBI形成胶质瘢痕提供了研究客体。
结论：用自由落体撞击法建立脑损伤胶质瘢痕形成模型,脑损伤确切,具备胶质瘢痕形成的条件。
第二部分大鼠脑损伤后胶质瘢痕的形态学确认
目的：观察星形胶质细胞聚集,并初步确定异常组织胶质瘢痕的形成及其范围。
方法：自由落体撞击法后,将脑损伤的大鼠饲养1周以上,以予损伤脑组织的瘢痕组织充分生长。分别在大鼠TBI后的第1周、2周及4周三个时间点灌注固定并取出大鼠脑组织,病理学检测,确定异常的胶质瘢痕组织。
结果：在三个时间点,各组均能清晰呈示出损伤点处的星形胶质细胞异常聚集,与正常脑组织之间边界清晰,且在TBI后第2周及4周的模型对照组鼠标本中,星形胶质细胞的异常聚集程度较1周更加明显,但2周和4周时间点之间的胶质细胞聚集程度没有明显差异变化。相对于模型对照组而言,三个治疗组的星形胶质细胞异常聚集明显减少。
结论：本实验条件下,大鼠TBI后的第1周,即出现胶质瘢痕；受损伤的脑组织与正常脑组织之间在星形胶质细胞聚集程度上差别明显。
第三部分硫酸软骨素酶ABC对脑损伤模型胶质瘢痕的影响
目的：比较观察ChABC对脑损伤胶质瘢痕的影响。
方法：各组标本根据免疫组化和蛋白印迹的不同要求,分别行4%多聚甲醛灌注和冰冻保存。其中多聚甲醛灌注后的标本经石蜡包埋、切片、脱蜡、及常规免疫组化检测CSPGs、GFAP分布情况；而用于检测蛋白印迹的冰冻标本,则经过蛋白提取和变性、胶的制作、点样、电泳、转膜、封闭、ECL检测,暗室曝光等操作后,观察CSPGs、GFAP表达情况。
结果：模型对照组CSPGs免疫组化染色后的脑损伤组织切片上,可清晰观察到胶质瘢痕组织呈淡黄色,胶质组织较正常脑组织染色深,边界清晰。而治疗组CSPGs免疫组化染色后的脑损伤切片,则淡黄色阳性结构较模型对照组明显减少,与正常组织边界模糊,其中以第3治疗组(5U/m1)最为明显。蛋白印迹的的检测提示5U/ml治疗组CSPGs表达较其它治疗组及模型对照组明显减少。
结论：经chABC治疗后的脑损伤在CSPGs的表达方面较未治疗组明显减少,且以5U/ml治疗组CSPGs表达减少最为明显。
Brain traumatic injury (TBI) is recognized as one of the injuries, with high morbidity, high cost, high mortality rate By using different ways of the treatment, there are 75% of survivors to lose their labor ability, and 40% of them with severe disability。The stats bring the heavy burden to both of family and society。There is still no satisfacyory stratergy to treat the TBI until now。In recent years, a series of experimental researches on nerve cells regeneration made the hopeness for TBI treatment。It was well know some of aspects about brain tissue regeneration as below:First, it is difficult for the nerve cells themselves to regenerate follwing the TBI。Secondly, oligodendrocytes can regenerate, but will form some of glial scar to obstruct the axonal regeneration。Thridly, the ability of axonal regeneration following the brain injury is influenced by many factors such as the inhibit factory and glail scar, but the axonal regeneration in CNS is still possible as soon as removing the inhibit factories in the damaged brain。Many new methods such as neural transplantations including peripheral nerve transplantation, NSCs transplantation, olfactory ensheathing cells transplant, gene therapy etc, can make adult animals to show some degree of recovery。
A series of reactions including the glail scar formation will start following the brain damage。The dense scar tissue will separate the normal brain tissue from the damaged area, and obstruct the renewable axons to generation by the glial scar。Meanwhile, the reactive astrocytes will secrete some of proteins polysaccharides during their accumulation, which also will inhibit the nerve regeneration in the injury brain。
It was proved that the chondroitin sulfate proteoglycans (CSPGs) possese the strong inhibiting rolo for axonal regeneration。However, the chondroitin sulfateABC (ChABC), as a kind of cleavage enzyme, can effectively reset the glail scar by degradeting the CSPGs.
This research will mainly include the contents as below。To establish the brain glial scar animal model after rat TBI, by freefall bump method。In treatment group, the different concentrations of ChABC were infected at the same time with TBI, and then the brain tissue specimens were harvested at the 1st,2nd and 4th week, following the TBI for observating the degree of the astrocytes accumulation, in order to understand the scar tissue growth situation。The positive expressions of CSPGs and glial fibers acidic acid protein (GFAP) were tested in order to estimating the scar healing.
Part One Rats glial scar model after TBI
Objective:To construct the glial scar model after rat TBI, and to supply the foundation for studing glail scar treatment further。
Methods:Thirty eight of wistar rats were divided into 5 groups inclueling: control group (n=9), treatment group 1～3 (n=9, respectively), and normal group (n=2)。Rats TBI model was made by freefall bump method。Hammer weighing 20 g and drop height 30 cm, so the impact was 20×30g·cm。Rat's head was fixed to the stereo-frame。Along The midline of the scalp, to cut skin about 2 cm, then to separate the cut subcutaneous fascia layer by layer, up to the skull, Around anterior fontanelle 2 mm and left 2 mm of sagittal seam on the left side of the brain, a circular with diameter 5mm was opened, but the dura mater remained intact。20g weightfalled on the epidural bone window from 30cm high, and then sutured scalp after hemostasis。Treatment groups (group 1～3) were treated by ChABC with different concentrations。
Results:The rat TBI model by freefall bump method provided research receptor for forming glial scar after rat TBI。
Conclusion:Using free fallers bump method to establish rat TBI model, the brain damage exactly, have the conditions for glial scar formation。
Part two Morphology of glial scar formation after the rat TBI
Objective:Observing the astrocytes aggregation and definiting the formation of the glial scar and its scope preliminarily。
Methods:Following the TBI by freefall bump method, the model rats were reared for much more than 1 week, in order to form glial scar in the injured brain tissue。The TBI'rats were fixed by perfussion with parafor and then the rats brains were removed respectively at the 1st,2nd and 4th week after TBI。The pathology was tested to determine the abnormal glial scar tissue。
Results:In three time points, abnorma astrocytel could be found in each group, with the clear border from the normal brain tissue。The abnormal aggregation of astrocytes showed much more obvious in the model control group at the 2nd week and 4th week after TBI than at the 1st week。However, there was no difference between the 2nd week and 4th week。Compared to the control group, the abnormal astrocytes aggregation in the three of treatment groups showed much less significantly。
Conclusion:The glia scar appeared just at the 1st week afer rat TBI。About the abnormal astrocytes aggregation, there was a significant difference between the normal and the injured brain tissue。
Part three The effect of ChABC on glial scar in the rat TBI model。
Objective:To observate the effect of ChABC on glial scar in the TBI rat。
Methods:The specimens in different groups were performed by 4% formaldehyde perfusion or reservation at freezing according to the different requirements of immunohistochemical or protein assay by western blot respectively。The specimens for the immunohistochemistry were detected the expression of CSPGs, GFAP respectively after paraffin embedding, sectioning, dewaxing and immunohistochemical test。On the other hand, The specimen for the western blot were observed the protein expression of CSPGs or GFAP respectively, after protein denaturation, glue production, electrophoresis, turn film, closed, ECL detection, and darkroom exposure, et。
Results:The CSPGs-positive expressing glial scar tissue showed the dark yellow in the model control group, but pale yellow in the treated groups。Amoung the three of treatment groups, the (5U/ml)ChABC treatment group showen the most obvious effect。The result of the western blot assay supported the immunohistochemical foundation。
Conclusion:ChABC reduced the expression of CSPGs in the treatment groups, compared with control group, especially 5U/ml group as the most obvious effect in this experiment。
脑损伤后会启动一系列与形成瘢痕组织相关的细胞及分子间的反应,形成以星形胶质细胞为主的瘢痕组织,这种致密的胶质瘢痕包囊脑的损伤区从而把脑损伤区与正常脑组织隔开,使再生的神经轴突不能越过损伤区的胶质瘢痕,造成再生的神经轴突不能与突触后神经元建立功能性链接；同时星形胶质细胞分泌的蛋白多糖即硫酸软骨素蛋白多糖(chondroitin sulfate proteoglycans, CSPGs)在损伤区的堆积也是抑制神经再生的重要原因。所以,胶质瘢痕组织是导致脑组织神经难以再生的主要原因之一。
研究证实,CSPGs有非常强的抑制轴突再生功能,而作为细菌胞外裂解酶的硫酸软骨素酶ABC (chondroitin sulfate enzyme ABC, chABC)则可以通过干预CSPGs对神经修复的抑制作用而减少神经胶质瘢痕,促进神经再生。
本课题研究主要内容是：使用自由落体撞击法建立脑损伤胶质瘢痕的动物模型,并依照分组条件在治疗组的动物损伤点上即时注射不同浓度的chABC;在此后的1周、2周及4周三个时间点取出各组脑组织标本,行镜下比较观察各组(包括正常组、模型组对照组、治疗组)的损伤点星形胶质细胞聚集程度以了解瘢痕组织生长情况,并以免疫组化和蛋白印迹观察CSPGs及胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)的表达情况,以判断去瘢痕的治疗效果。
第一部分大鼠脑损伤后胶质瘢痕模型的制作
目的：构建大鼠脑损伤胶质瘢痕模型,为后续进一步对去瘢痕组织治疗研究奠定基础。
方法：wistar大鼠随机分为4组,每组9只,分别为模型对照组、治疗组1(1U/ml ChABC)、治疗组2(2.5U/ml ChABC)、治疗组3(5U/ml ChABC);另还有2只为正常对照组。采用改进的Feeney法按自由落体原理制造成大鼠重度TBI模型。撞击锤重20 g,下落高度30 cm,落体致伤的冲击力为20×30 g·cm。将大鼠头部固定于立体头架上,沿头皮中线矢状位切开皮肤约2 cm,然后逐层切开皮下筋膜,分离骨膜至颅骨,以前囱尾侧2mm、矢状缝左侧2mm的左侧大脑皮质后肢运动区域为中心,开一直径为5mm的圆形骨窗,硬膜保持完整。20 g撞击捶从30cm高的外周套管下落,撞击已置于骨窗硬膜上的撞杆；撞击伤后,充分止血即缝合头皮。治疗组则即时在损伤点注射不同浓度的chABC。
结果：以Feeney自由落体撞击法建立了重度大鼠TBI模型,为大鼠TBI形成胶质瘢痕提供了研究客体。
结论：用自由落体撞击法建立脑损伤胶质瘢痕形成模型,脑损伤确切,具备胶质瘢痕形成的条件。
第二部分大鼠脑损伤后胶质瘢痕的形态学确认
目的：观察星形胶质细胞聚集,并初步确定异常组织胶质瘢痕的形成及其范围。
方法：自由落体撞击法后,将脑损伤的大鼠饲养1周以上,以予损伤脑组织的瘢痕组织充分生长。分别在大鼠TBI后的第1周、2周及4周三个时间点灌注固定并取出大鼠脑组织,病理学检测,确定异常的胶质瘢痕组织。
结果：在三个时间点,各组均能清晰呈示出损伤点处的星形胶质细胞异常聚集,与正常脑组织之间边界清晰,且在TBI后第2周及4周的模型对照组鼠标本中,星形胶质细胞的异常聚集程度较1周更加明显,但2周和4周时间点之间的胶质细胞聚集程度没有明显差异变化。相对于模型对照组而言,三个治疗组的星形胶质细胞异常聚集明显减少。
结论：本实验条件下,大鼠TBI后的第1周,即出现胶质瘢痕；受损伤的脑组织与正常脑组织之间在星形胶质细胞聚集程度上差别明显。
第三部分硫酸软骨素酶ABC对脑损伤模型胶质瘢痕的影响
目的：比较观察ChABC对脑损伤胶质瘢痕的影响。
方法：各组标本根据免疫组化和蛋白印迹的不同要求,分别行4%多聚甲醛灌注和冰冻保存。其中多聚甲醛灌注后的标本经石蜡包埋、切片、脱蜡、及常规免疫组化检测CSPGs、GFAP分布情况；而用于检测蛋白印迹的冰冻标本,则经过蛋白提取和变性、胶的制作、点样、电泳、转膜、封闭、ECL检测,暗室曝光等操作后,观察CSPGs、GFAP表达情况。
结果：模型对照组CSPGs免疫组化染色后的脑损伤组织切片上,可清晰观察到胶质瘢痕组织呈淡黄色,胶质组织较正常脑组织染色深,边界清晰。而治疗组CSPGs免疫组化染色后的脑损伤切片,则淡黄色阳性结构较模型对照组明显减少,与正常组织边界模糊,其中以第3治疗组(5U/m1)最为明显。蛋白印迹的的检测提示5U/ml治疗组CSPGs表达较其它治疗组及模型对照组明显减少。
结论：经chABC治疗后的脑损伤在CSPGs的表达方面较未治疗组明显减少,且以5U/ml治疗组CSPGs表达减少最为明显。
Brain traumatic injury (TBI) is recognized as one of the injuries, with high morbidity, high cost, high mortality rate By using different ways of the treatment, there are 75% of survivors to lose their labor ability, and 40% of them with severe disability。The stats bring the heavy burden to both of family and society。There is still no satisfacyory stratergy to treat the TBI until now。In recent years, a series of experimental researches on nerve cells regeneration made the hopeness for TBI treatment。It was well know some of aspects about brain tissue regeneration as below:First, it is difficult for the nerve cells themselves to regenerate follwing the TBI。Secondly, oligodendrocytes can regenerate, but will form some of glial scar to obstruct the axonal regeneration。Thridly, the ability of axonal regeneration following the brain injury is influenced by many factors such as the inhibit factory and glail scar, but the axonal regeneration in CNS is still possible as soon as removing the inhibit factories in the damaged brain。Many new methods such as neural transplantations including peripheral nerve transplantation, NSCs transplantation, olfactory ensheathing cells transplant, gene therapy etc, can make adult animals to show some degree of recovery。
A series of reactions including the glail scar formation will start following the brain damage。The dense scar tissue will separate the normal brain tissue from the damaged area, and obstruct the renewable axons to generation by the glial scar。Meanwhile, the reactive astrocytes will secrete some of proteins polysaccharides during their accumulation, which also will inhibit the nerve regeneration in the injury brain。
It was proved that the chondroitin sulfate proteoglycans (CSPGs) possese the strong inhibiting rolo for axonal regeneration。However, the chondroitin sulfateABC (ChABC), as a kind of cleavage enzyme, can effectively reset the glail scar by degradeting the CSPGs.
This research will mainly include the contents as below。To establish the brain glial scar animal model after rat TBI, by freefall bump method。In treatment group, the different concentrations of ChABC were infected at the same time with TBI, and then the brain tissue specimens were harvested at the 1st,2nd and 4th week, following the TBI for observating the degree of the astrocytes accumulation, in order to understand the scar tissue growth situation。The positive expressions of CSPGs and glial fibers acidic acid protein (GFAP) were tested in order to estimating the scar healing.
Part One Rats glial scar model after TBI
Objective:To construct the glial scar model after rat TBI, and to supply the foundation for studing glail scar treatment further。
Methods:Thirty eight of wistar rats were divided into 5 groups inclueling: control group (n=9), treatment group 1～3 (n=9, respectively), and normal group (n=2)。Rats TBI model was made by freefall bump method。Hammer weighing 20 g and drop height 30 cm, so the impact was 20×30g·cm。Rat's head was fixed to the stereo-frame。Along The midline of the scalp, to cut skin about 2 cm, then to separate the cut subcutaneous fascia layer by layer, up to the skull, Around anterior fontanelle 2 mm and left 2 mm of sagittal seam on the left side of the brain, a circular with diameter 5mm was opened, but the dura mater remained intact。20g weightfalled on the epidural bone window from 30cm high, and then sutured scalp after hemostasis。Treatment groups (group 1～3) were treated by ChABC with different concentrations。
Results:The rat TBI model by freefall bump method provided research receptor for forming glial scar after rat TBI。
Conclusion:Using free fallers bump method to establish rat TBI model, the brain damage exactly, have the conditions for glial scar formation。
Part two Morphology of glial scar formation after the rat TBI
Objective:Observing the astrocytes aggregation and definiting the formation of the glial scar and its scope preliminarily。
Methods:Following the TBI by freefall bump method, the model rats were reared for much more than 1 week, in order to form glial scar in the injured brain tissue。The TBI'rats were fixed by perfussion with parafor and then the rats brains were removed respectively at the 1st,2nd and 4th week after TBI。The pathology was tested to determine the abnormal glial scar tissue。
Results:In three time points, abnorma astrocytel could be found in each group, with the clear border from the normal brain tissue。The abnormal aggregation of astrocytes showed much more obvious in the model control group at the 2nd week and 4th week after TBI than at the 1st week。However, there was no difference between the 2nd week and 4th week。Compared to the control group, the abnormal astrocytes aggregation in the three of treatment groups showed much less significantly。
Conclusion:The glia scar appeared just at the 1st week afer rat TBI。About the abnormal astrocytes aggregation, there was a significant difference between the normal and the injured brain tissue。
Part three The effect of ChABC on glial scar in the rat TBI model。
Objective:To observate the effect of ChABC on glial scar in the TBI rat。
Methods:The specimens in different groups were performed by 4% formaldehyde perfusion or reservation at freezing according to the different requirements of immunohistochemical or protein assay by western blot respectively。The specimens for the immunohistochemistry were detected the expression of CSPGs, GFAP respectively after paraffin embedding, sectioning, dewaxing and immunohistochemical test。On the other hand, The specimen for the western blot were observed the protein expression of CSPGs or GFAP respectively, after protein denaturation, glue production, electrophoresis, turn film, closed, ECL detection, and darkroom exposure, et。
Results:The CSPGs-positive expressing glial scar tissue showed the dark yellow in the model control group, but pale yellow in the treated groups。Amoung the three of treatment groups, the (5U/ml)ChABC treatment group showen the most obvious effect。The result of the western blot assay supported the immunohistochemical foundation。
Conclusion:ChABC reduced the expression of CSPGs in the treatment groups, compared with control group, especially 5U/ml group as the most obvious effect in this experiment。
引文
[1]许刚,徐如祥,姜晓丹,周德祥,姚鹏飞,秦昆,蔡颖谦,邹雨汐,秦玲莎.绿色荧光蛋白基因腺病毒载体转染大鼠骨髓间充质干细胞的实验研究[J].中华神经医学杂志,2008,11(6)：153-156.
[2]Liesi P, Kauppila T.Induction of type IV collagen and other basement-membrane-associated proteins after spinal cord injury of the adult rat may participate in formation of the glial scar[J].Exp Neurol,2002,173 (1):31-45.
[3]Properzi F, Asher RA, Fawcelt JW.Chondroitin sulphate proteoglycans in the centeal nervous system:change and synthesis after injury[J].Biochem Soc Trans,2003,2 (6):335-336.
[4]Rhode KE, Moon LD, Fawcelt JW.Inhibiting cell proliferation during formation of the glial scar:effect on axon regeneration in the CNS[J].Neuroscience,2003,1 (7):41-56.
[5]Fouad K,Dietz V,Schwab ME.Improving axonal growth and functional recovery after experimental spinal cord injury by neutralizing myelin associated inhibitors [J].Brain Res Rev,2001,36 (2-3):204-212.
[6]Huang DW,McKerracher L,Braun PE,et al.A therapeutic vaccine approach to stimulate axon regeneration in the adult mammalian spinal cord [J].Neuron,1999,24(3):639-647.
[7]Lin R, Kwok JC, Crespo D, et al. Chondroitinase ABC has a long2lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain. J Neurochem,2008,104(2):1425-1428.
[8]Barritt AW, Davies M, Marchand F, et al.ChondroitinaseABC promotes sprouting of intact and injured spinal systems after spinal cord injury[J].J Neurosci,2006,42:10856-10867.
[9]William BIC, Shih-Hung Y, Pilip JD, et al.Functional axonal regeneration through astrocytic scar genetically modified to digest chondroitin sulfate proteoglycans[J].J Neurosci,2007,9 (7):2176-2185.
[10]McKeon RJ, Hoke A, Silver J. Injury2induced p roteoglycans inhibit the potential for laminin 2mediated axon growth on as2 trocytic scars. Exp Neurol, 1995,136(1):32-24.
[11]Massey JM, Hubscher CH, Wagoner MR, et al. Chondroiti2 nase ABC digestion of the perineuronal net p romotes functional collateral sp routing in the cuneate nucleus after cervical sp inal cord injury. J Neurosci,2006 26(16):1465-1469.
[12]Monnier PP, Sierra A, Schwab JM, et al. The Rho/ROCK pathway mediates neurite growth 2inhibitory activity associated with the chondroitin sulfate p roteoglycans of the CNS glial scar. Mol Cell Neurosci,2003,22 (3):318-321.
[13]Zuo J, Neubaner D, Graham J, et al. Regeneration of axons after nerve transaction repair is enhanced by degradation of chondroitin sulfate p roteoglycan. Exp Neurol,2002,176(1):221-222.
[14]徐云强,冯世庆,王沛,马信龙,宁广智,班德翔。硫酸软骨素酶促进大鼠脊髓损伤后轴突再生。中国矫形外科杂志,2010,18(7)：98-102.
[15]Rachel Lin, Jessica C. F. Kwok, Damaso Crespo_ and James W. Fawcett Chondroitinase ABC has a long-lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain J Neuro chew,2008,104 (9):400-408.
[16]Huang WC, Kuo WC, Cherng JH et al, Chondroitinase ABC promotes axonal re-growth and behavior recovery in spinal cord injury。Biochem Biophy Res Commu 2010,349 (12):963-968.
[17]柴宏伟；硫酸软骨素酶ABC治疗脊髓损伤时间窗的研究[D]；天津医科大学学报,2010,11(4)：128-132.
[1]卢奕,吴智远,惠国桢,郭礼和。暨荀鹤。一种症状性大鼠脑损伤模型的建立,苏州大学医学科学杂志。2005,25(5)：98-101.
[2]Silver J, Miler JH。Regeneration beyond glial scar[J]。Nat Rev Neurosci,2004, 5 (2):146-156.
[3]Jung C, Lilly YW, et al, Matrix Metalloproteinase-9 Facilitates Glial Scar Formation in the Injured Spinal Cord [J]. The Journal of Neuroscience,2008, 28(50):13467-13477.
[4]Deng C, Gorrie C, Hayward I, et al。Survival and migration of human and rat olfactory ensheathing cells in intact and injured spinal cord. J Neurosc Res, 2006,83(7):1201-1212.
[5]Keyvan-Fouladi N, Raisman G, Li Y。Delayed repair of corticospinal tract lesions as an assay for the effectiveness of transplantation of Schwann cells。 Glia,2005,51(4):306-311.
[6]韩玉河,吕然博,张万宏,等。脑内注射人胚胎鞘细胞对颅脑损伤大鼠神经及运动功能缺失的影响.中国临床康复,2005,9(5)：229-231.
[7]徐云强,冯世庆,王沛,等。硫酸软骨素酶促进大鼠脊髓损伤后轴突再生。中国矫形外科杂志,2010,18(7)210-213.
[1]Fouad K, Dietz V, Schwab ME.Improving axonal growth and functional recovery after experimental spinal cord injury by neutralizing myelin associated inhibitors [J]。Brain Res Rev,2001,36 (2-3):204-212.
[2]Huang DW, McKerracher L, Braun PE, et al.A therapeutic vaccine approach to stimulate axon regeneration in the adult mammalian spinal cord [J]。 Neuron,1999,24(3)::639-647.
[3]朱舟,王伟,谢明杰,等。CDK抑制剂olomoucine对星形胶质细胞增值活化的影响[J].华中科技大学学报,2005,34(2)：159-163.
[4]Hobhm C, Gunther A, Grosche J, etal.Decomposition and long—lasting downregulation of extracellular matrix in perineuronal nets induced by focalcerebral ischemia in rats[J].J Neurosci Res,2005,80(4):539-548.
[5]Kozuka N, Itofusa R, Kudo Y, et al.Lipopolysaccharide and proinflammatory cytokines require different astrocyte states to induce nitric oxide production [J].J Neurosci Res,2005,82 (5):717-728.
[6]Alonso K。G.NG2 proteoglycan-expressing cells of the adult rat brain: possible involvement in the formation of glial scar astrocytes following stab wound[J].Glia,2005,49 (3):318-338.
[7]Culmsee C, Strumm RK, Schafer M K, et al. Clenbuterol induce growth factor Mrna,activates astrocytes, and protects rat brain tissue against ischemic damage[J]。Eur J Pharmacol,1999,379 (1):33-45.
[8]Gimenez Y, Ribotta M, Menet V, et al。Tenascin C induces a quiescent phenotype in cultured adult human astrocytes[J]。Glia,2001,132(5):587-610.
[9]李建明,初同伟。脊髓损伤后星形胶质细胞的病理变化及相关治疗措施进展。中国脊柱脊髓杂志[J],2007,17(8)：632-634.
[10]李兰,林江凯。脊髓损伤后胶质瘢痕形成中补体的作用。创伤外科杂志[J],2010,6(3)：110-113.
[11]Lin JK, Cai WQ。Effect of vimentin on reactive gliosis:in vitro and in vivo analysis [J]。J Neurotra,2004,21(11):1671-1682.
[12]Silver J, Miller, JH。Regeneration beyond the glial scar [J]。Nat Rev Nerosci, 2004,5(2):146-156.
[13]Alonso G.. N G2 proteoglycan-expressing cells of the adult rat brain:possible involvement in the formation of glai scar astrocytes following stab wound [J]。 Glia,2008,49 (3):318-338.
[14]West H, Richardson WD, Fruttiger M. Stabilization of the retinal vascular network by reciprocal feedback between blood vessels and astrocytes。 Development [J],2009,132 (8):1855-1862.
[1]Liesi P, Kauppila T。Induction of type IV collagen and other basement-membrane-associated proteins after spinal cord injury of the adult rat may participate in formation of the glial scar[J]。Exp Neurol,2002,173 (1): 31-45.
[2]苏国辉。拮抗瘢痕组织障碍神经再生的作用[J]。中国处方药,2004,2(10)：31-33.
[3]Monnier PP, Sierra A, Macchi P, et al. RGM is a repulsive guidance molecule for retinal axons [J]。Nature,2002,419(6905):392-395。
[4]Borisoff JF, Chan CC, Hiebert GW, et al。Suppression of Rhokinase activity promotes axonal growth on inhibitory CNS substrates [J].Mol Cell Neurosci, 2003,22 (3):405-416.
[5]何华。如何正确地对免疫组化结果进行半定量评分。中外健康文摘·临床医师,2008,5(7)：162-163.
[6]Mi S, Lee X, shao Z, et al。LINGO-1 is a component of the Nogo-66receptor/p75 Signaling complex。Nat Neurosci,2006,7(3):221-228.
[7]Silver J, Miler JH。Regeneration beyond glial scar.Nat Rev Neurosci,2004, 5 (2):146-156.
[8]Garulli D, Laabs T, Geller HM, et al。Chondroitin sulfate proteoglycans in neural development and regeneration[J].Curr Opin Nerobiol,2005,15 (2): 116-120.
[9]Iaci JF, Vecchione AM, Zimber MP, et al。Chondroitin sulfate proteoglycans in spinal cord contusion injury and the effects of Chondroitinase treatment [J]。 J Neurotrauma,2009,24 (14):1743-1759.
[10]Yick LW, Cheung PT, So K, et al.Axonal regeneration of Clarke's neurons beyond the spinal cord injury scar after treatment with ChondroitinaseABC [J]。Exp Neurol,2003,182 (1):160-168.
[11]赵伟,张连双。大鼠脊髓半横断损伤后硫酸软骨素蛋白多糖Aggrecan的变化。滨州医学院学报,2010,33(5)：325-327.
[12]谢群,林扬元,陈建明。硫酸软骨素酶ABC对大鼠脊髓损伤后硫酸软骨素蛋白多糖与胶质纤维酸性蛋白表达的影响。新乡医学院学报,2010,27(5)：51-53.
[1]Aguayo AJ, David S, Bray GM.Influences of the glial environment on the elongation of axons after injury transplantation studies in adult rodents. Journal of Experimental Biology,1981,95(3):231-240.
[2]唐洲平,郭守刚,康慧聪,等。嗅鞘细胞移植治疗脑出血的实验研究.中国现代医学杂志,2005,15(11)：1628-1630.
[3]Ramer LM, Richter MW, Roskams AJ, et al. Peripherally-derived olfactory ensheathing cells do not promote primary afferent.regeneration following dorsal root injury.Glia.2004,47(2):189-206.
[4]Ramon-Cueto A, Plant GW, Avila J, et al. Long distance axonal regeneration in the transected adult rat spinal cord is promoted by olfactory ensheathing glia transplants. J Neurosci,1998,18 (1):3803-3815.
[5]Bradbury EJ, Moon LDF, Popat RJ, et al. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature,2002,416(6881):636-640.
[6]Roll A, Avidan H, Cahalon L, et al. A disaccharide derived from chondroitin sulphate proteoglycan promotes central nervous system repair in rats and mice. Eur J Neurosci,2004,20(8):1973-1983.
[7]林扬元,谢群,陈建明,等。硫酸软骨素酶ABC对大鼠脊髓损伤修复的影响.神经解剖学杂志,2007,23(3)：288-292.
[8]赵伟,张连双。大鼠脊髓半横断损伤后硫酸软骨素蛋白多糖Aggrecan的 变化,滨州医学院学报2010,33(5)：58-61.
[9]谢群,林杨元,陈建明。硫酸软骨素酶ABC对大鼠脊髓损伤后硫酸软骨素蛋白多糖与胶质纤维酸性蛋白表达的影响。新乡医学院学报,2010,27(5)：51-53.
[10]傅佳峰,诸葛启钏,周盛轩。嗅鞘细胞移植对改善大鼠脑出血后神经功能缺损的研究。中华实验外科杂志,2007,24(3)：374.
[11]黄红云,王洪美,修波,等.嗅鞘细胞移植治疗脊髓损伤临床试验的初步报告,海军总医院学报,2002,15(1)：18-21.
[12]徐云强,冯世庆,王沛,等。硫酸软骨素酶促进大鼠脊髓损伤后轴突再生。中国矫形外科杂志,2010,18(7)210-213.
[13]柴宏伟；硫酸软骨素酶ABC治疗脊髓损伤时间窗的研究[D]；天津医科大学学报,2009,11(4)：128-132.
[14]Rachel Lin, Jessica C, Damaso Crespo and James W. Fawcett, Chondroitinase ABC has a long-lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain, N eurochem,2008,104 (6):400-408.
[15]Deng C, Gorrie C, Hayward I, et al。Survival and migration of human and rat olfactory ensheathing cells in intact and injured spinal cord. J Neurosci Res, 2010,83(7):1201-1212.
[16]Keyvan-Fouladi N, Raisman G, Li Y. Delayed repair of corticospinal tract lesions as an assay for the effectiveness of transplantation of Schwann cells.Glia,2009,51(4):306-311.
[17]韩玉河,吕然博,张万宏,等。脑内注射人胚胎鞘细胞对颅脑损伤大鼠神经及运动功能缺失的影响。中国临床康复,2005,9(5)：229-231.
[2]Liesi P, Kauppila T.Induction of type IV collagen and other basement-membrane-associated proteins after spinal cord injury of the adult rat may participate in formation of the glial scar[J].Exp Neurol,2002,173 (1):31-45.
[3]Properzi F, Asher RA, Fawcelt JW.Chondroitin sulphate proteoglycans in the centeal nervous system:change and synthesis after injury[J].Biochem Soc Trans,2003,2 (6):335-336.
[4]Rhode KE, Moon LD, Fawcelt JW.Inhibiting cell proliferation during formation of the glial scar:effect on axon regeneration in the CNS[J].Neuroscience,2003,1 (7):41-56.
[5]Fouad K,Dietz V,Schwab ME.Improving axonal growth and functional recovery after experimental spinal cord injury by neutralizing myelin associated inhibitors [J].Brain Res Rev,2001,36 (2-3):204-212.
[6]Huang DW,McKerracher L,Braun PE,et al.A therapeutic vaccine approach to stimulate axon regeneration in the adult mammalian spinal cord [J].Neuron,1999,24(3):639-647.
[7]Lin R, Kwok JC, Crespo D, et al. Chondroitinase ABC has a long2lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain. J Neurochem,2008,104(2):1425-1428.
[8]Barritt AW, Davies M, Marchand F, et al.ChondroitinaseABC promotes sprouting of intact and injured spinal systems after spinal cord injury[J].J Neurosci,2006,42:10856-10867.
[9]William BIC, Shih-Hung Y, Pilip JD, et al.Functional axonal regeneration through astrocytic scar genetically modified to digest chondroitin sulfate proteoglycans[J].J Neurosci,2007,9 (7):2176-2185.
[10]McKeon RJ, Hoke A, Silver J. Injury2induced p roteoglycans inhibit the potential for laminin 2mediated axon growth on as2 trocytic scars. Exp Neurol, 1995,136(1):32-24.
[11]Massey JM, Hubscher CH, Wagoner MR, et al. Chondroiti2 nase ABC digestion of the perineuronal net p romotes functional collateral sp routing in the cuneate nucleus after cervical sp inal cord injury. J Neurosci,2006 26(16):1465-1469.
[12]Monnier PP, Sierra A, Schwab JM, et al. The Rho/ROCK pathway mediates neurite growth 2inhibitory activity associated with the chondroitin sulfate p roteoglycans of the CNS glial scar. Mol Cell Neurosci,2003,22 (3):318-321.
[13]Zuo J, Neubaner D, Graham J, et al. Regeneration of axons after nerve transaction repair is enhanced by degradation of chondroitin sulfate p roteoglycan. Exp Neurol,2002,176(1):221-222.
[14]徐云强,冯世庆,王沛,马信龙,宁广智,班德翔。硫酸软骨素酶促进大鼠脊髓损伤后轴突再生。中国矫形外科杂志,2010,18(7)：98-102.
[15]Rachel Lin, Jessica C. F. Kwok, Damaso Crespo_ and James W. Fawcett Chondroitinase ABC has a long-lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain J Neuro chew,2008,104 (9):400-408.
[16]Huang WC, Kuo WC, Cherng JH et al, Chondroitinase ABC promotes axonal re-growth and behavior recovery in spinal cord injury。Biochem Biophy Res Commu 2010,349 (12):963-968.
[17]柴宏伟；硫酸软骨素酶ABC治疗脊髓损伤时间窗的研究[D]；天津医科大学学报,2010,11(4)：128-132.
[1]卢奕,吴智远,惠国桢,郭礼和。暨荀鹤。一种症状性大鼠脑损伤模型的建立,苏州大学医学科学杂志。2005,25(5)：98-101.
[2]Silver J, Miler JH。Regeneration beyond glial scar[J]。Nat Rev Neurosci,2004, 5 (2):146-156.
[3]Jung C, Lilly YW, et al, Matrix Metalloproteinase-9 Facilitates Glial Scar Formation in the Injured Spinal Cord [J]. The Journal of Neuroscience,2008, 28(50):13467-13477.
[4]Deng C, Gorrie C, Hayward I, et al。Survival and migration of human and rat olfactory ensheathing cells in intact and injured spinal cord. J Neurosc Res, 2006,83(7):1201-1212.
[5]Keyvan-Fouladi N, Raisman G, Li Y。Delayed repair of corticospinal tract lesions as an assay for the effectiveness of transplantation of Schwann cells。 Glia,2005,51(4):306-311.
[6]韩玉河,吕然博,张万宏,等。脑内注射人胚胎鞘细胞对颅脑损伤大鼠神经及运动功能缺失的影响.中国临床康复,2005,9(5)：229-231.
[7]徐云强,冯世庆,王沛,等。硫酸软骨素酶促进大鼠脊髓损伤后轴突再生。中国矫形外科杂志,2010,18(7)210-213.
[1]Fouad K, Dietz V, Schwab ME.Improving axonal growth and functional recovery after experimental spinal cord injury by neutralizing myelin associated inhibitors [J]。Brain Res Rev,2001,36 (2-3):204-212.
[2]Huang DW, McKerracher L, Braun PE, et al.A therapeutic vaccine approach to stimulate axon regeneration in the adult mammalian spinal cord [J]。 Neuron,1999,24(3)::639-647.
[3]朱舟,王伟,谢明杰,等。CDK抑制剂olomoucine对星形胶质细胞增值活化的影响[J].华中科技大学学报,2005,34(2)：159-163.
[4]Hobhm C, Gunther A, Grosche J, etal.Decomposition and long—lasting downregulation of extracellular matrix in perineuronal nets induced by focalcerebral ischemia in rats[J].J Neurosci Res,2005,80(4):539-548.
[5]Kozuka N, Itofusa R, Kudo Y, et al.Lipopolysaccharide and proinflammatory cytokines require different astrocyte states to induce nitric oxide production [J].J Neurosci Res,2005,82 (5):717-728.
[6]Alonso K。G.NG2 proteoglycan-expressing cells of the adult rat brain: possible involvement in the formation of glial scar astrocytes following stab wound[J].Glia,2005,49 (3):318-338.
[7]Culmsee C, Strumm RK, Schafer M K, et al. Clenbuterol induce growth factor Mrna,activates astrocytes, and protects rat brain tissue against ischemic damage[J]。Eur J Pharmacol,1999,379 (1):33-45.
[8]Gimenez Y, Ribotta M, Menet V, et al。Tenascin C induces a quiescent phenotype in cultured adult human astrocytes[J]。Glia,2001,132(5):587-610.
[9]李建明,初同伟。脊髓损伤后星形胶质细胞的病理变化及相关治疗措施进展。中国脊柱脊髓杂志[J],2007,17(8)：632-634.
[10]李兰,林江凯。脊髓损伤后胶质瘢痕形成中补体的作用。创伤外科杂志[J],2010,6(3)：110-113.
[11]Lin JK, Cai WQ。Effect of vimentin on reactive gliosis:in vitro and in vivo analysis [J]。J Neurotra,2004,21(11):1671-1682.
[12]Silver J, Miller, JH。Regeneration beyond the glial scar [J]。Nat Rev Nerosci, 2004,5(2):146-156.
[13]Alonso G.. N G2 proteoglycan-expressing cells of the adult rat brain:possible involvement in the formation of glai scar astrocytes following stab wound [J]。 Glia,2008,49 (3):318-338.
[14]West H, Richardson WD, Fruttiger M. Stabilization of the retinal vascular network by reciprocal feedback between blood vessels and astrocytes。 Development [J],2009,132 (8):1855-1862.
[1]Liesi P, Kauppila T。Induction of type IV collagen and other basement-membrane-associated proteins after spinal cord injury of the adult rat may participate in formation of the glial scar[J]。Exp Neurol,2002,173 (1): 31-45.
[2]苏国辉。拮抗瘢痕组织障碍神经再生的作用[J]。中国处方药,2004,2(10)：31-33.
[3]Monnier PP, Sierra A, Macchi P, et al. RGM is a repulsive guidance molecule for retinal axons [J]。Nature,2002,419(6905):392-395。
[4]Borisoff JF, Chan CC, Hiebert GW, et al。Suppression of Rhokinase activity promotes axonal growth on inhibitory CNS substrates [J].Mol Cell Neurosci, 2003,22 (3):405-416.
[5]何华。如何正确地对免疫组化结果进行半定量评分。中外健康文摘·临床医师,2008,5(7)：162-163.
[6]Mi S, Lee X, shao Z, et al。LINGO-1 is a component of the Nogo-66receptor/p75 Signaling complex。Nat Neurosci,2006,7(3):221-228.
[7]Silver J, Miler JH。Regeneration beyond glial scar.Nat Rev Neurosci,2004, 5 (2):146-156.
[8]Garulli D, Laabs T, Geller HM, et al。Chondroitin sulfate proteoglycans in neural development and regeneration[J].Curr Opin Nerobiol,2005,15 (2): 116-120.
[9]Iaci JF, Vecchione AM, Zimber MP, et al。Chondroitin sulfate proteoglycans in spinal cord contusion injury and the effects of Chondroitinase treatment [J]。 J Neurotrauma,2009,24 (14):1743-1759.
[10]Yick LW, Cheung PT, So K, et al.Axonal regeneration of Clarke's neurons beyond the spinal cord injury scar after treatment with ChondroitinaseABC [J]。Exp Neurol,2003,182 (1):160-168.
[11]赵伟,张连双。大鼠脊髓半横断损伤后硫酸软骨素蛋白多糖Aggrecan的变化。滨州医学院学报,2010,33(5)：325-327.
[12]谢群,林扬元,陈建明。硫酸软骨素酶ABC对大鼠脊髓损伤后硫酸软骨素蛋白多糖与胶质纤维酸性蛋白表达的影响。新乡医学院学报,2010,27(5)：51-53.
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