E3B1基因的表达调控与轴突再生的相关性研究
摘要
研究背景
CNS损伤是一种后果极为严重的创伤,由于疗效差且多发生于青壮年,对个人、家庭和社会都会带来巨大负担。如何促进CNS损伤后的再生修复及功能重建已成为当今骨外科学研究的热点和难点课题之一。
CNS损伤后,髓磷脂(myelin)等轴突生长抑制物的存在使神经元轴突回缩和崩溃,抑制了轴突的再生,也限制了CNS损伤后的修复。轴突生长抑制因子使轴突再生失败的作用机制,最终是通过使神经元细胞骨架肌动蛋白发生解聚、stress fiber形成、抑制细胞骨架重建而实现的。有研究证实启动神经元内肌动蛋白发生聚合,细胞骨架重建,可以促进轴突生长。因此,如何启动神经元内肌动蛋白发生聚合作用、促进神经元细胞骨架重建是解除抑制因子对轴突再生抑制、促进轴突定向生长、进而促进CNS损伤修复的关键所在。目前一般认为启动神经元内肌动蛋白发生聚合,促进微丝、微管细胞骨架重建,从而解除抑制因子对轴突再生的抑制,促进轴突定向生长,是一个多环节的、多种基因参与的过程。但在这一过程中可能存在着某个或某些关键性基因,从这些关键性基因着手,就会为解除抑制因子对轴突再生的抑制作用,促进轴突定向生长、进而促进CNS损伤修复治疗提供新的思路。
E3B1(也称为Abi1)于1995年作为调节细胞生长和增殖相关的非受体蛋白酪氨酸激酶Abl(也称为Eps8)的结合蛋白得到分离和鉴定[12]。其同源蛋白家族主要包括Abi2、Agbpl和NESH。进一步研究证实: E3B1在细胞骨架重建和细胞突起形成中发挥关键性作用,其过度表达能够引起肌动蛋白的解聚以及抑制细胞骨架重建。因此, E3B1可能是调控肌动蛋白聚合和细胞骨架重建的关键性基因。为此,本课题采用分子生物学技术,探讨神经元轴突抑制前后E3B1的表达变化规律,并通过RNA干扰(RNAi)技术,设计、构建、筛选和转染siRNA,抑制神经元内E3B1的表达,以促进轴突的再生。为轴突再生抑制的治疗提供新的思路,并为临床上促进CNS损伤后的再生修复探索新的途径。
研究内容及方法
1、E3B1基因在急性脊髓损伤大鼠脊髓组织中的表达变化研究建立大鼠急性脊髓损伤模型,采用免疫组织化学、RT-PCR、Western blot研究方法,动态观测大鼠脊髓损伤前后脊髓组织中E3B1蛋白和E3B1mRNA的表达变化,分析了E3B1基因表达变化在脊髓损伤中的意义。
2、大鼠皮层神经元的分离、培养、鉴定和CNS髓鞘质的提取、鉴定对大鼠大脑皮层组织进行原代分离培养,观察其生长和形态结构,并采用GFAP与NF200荧光标记,确认神经元的形态和数量。提取了CNS髓鞘质,观测不同浓度CNS髓鞘质对神经元轴突生长的影响,选取最适实验浓度,并采用Western blot进行成分鉴定。
3、神经元细胞E3B1基因的表达变化检测
1)采用免疫细胞化学观测了E3B1在培养神经元细胞中的表达分布,采用RT-PCR、Western blot检测培养神经元细胞中E3B1mRNA和E3B1蛋白的表达。
2)培养神经元细胞中加入提取的轴突生长抑制物CNS髓鞘质,采用RT-PCR、Western blot检测了神经元轴突抑制时E3B1蛋白和E3B1mRNA的表达变化。
3)采用免疫荧光标记对比观测了培养神经元细胞加入CNS髓鞘质前后, E3B1和?Ш-tubulin在神经元细胞中的表达变化,分析了E3B1基因表达变化与神经元轴突生长抑制的相关性。
4、E3B1基因的表达调控对培养神经元细胞轴突再生的影响
1)针对E3B1的RNA干扰质粒载体的构建:选择针对E3B1(NCBI:NM-024397)的RNAi靶位点三个(分别命名为Abi11,Abi12,Abi13)和已明确的不针对任何mRNA的RNAi靶位点一个(阴性对照,命名HK),以及阳性对照GAPDH-A,选用带有neoR选择标志和GFP绿色荧光标志的真核表达载体pGenesil-1构建E3B1RNAi质粒。并进行了抗性筛选、酶切、测序鉴定。
2) RNA干扰对神经元细胞E3B1表达的影响研究:将构建好的RNAi质粒载体通过Lipofectamine 2000依次转染培养的神经元细胞,采用荧光显微镜检测转染率,RT-PCR和Western blot检测针对E3B1不同靶位点的RNAi对神经元细胞E3B1基因表达的影响,并筛选出了对E3B1基因具有最佳抑制效应的siRNA。
3)调控E3B1表达对神经元轴突生长的影响:将筛选出的对E3B1基因具有最佳抑制效应的siRNA转染神经元细胞,经抗性筛选,加入轴突生长抑制物,采用?Ш-tubulin免疫荧光标记和Western blot对比观测E3B1基因表达抑制的神经元轴突的生长状况。
结果
1、E3B1基因在急性脊髓损伤大鼠脊髓组织中的表达变化研究
正常脊髓组织中存在少量E3B1 mRNA和蛋白表达,主要位于脊髓灰质区,急性脊髓损伤后6h E3B1 mRNA和蛋白的表达已明显升高,并于24-48h达高峰,之后逐渐降低。
2、大鼠皮层神经元的分离、培养、鉴定和CNS髓鞘质的提取、鉴定
神经元接种后约3h逐步有细胞开始贴壁, 7～10 d左右细胞形态和数量较为稳定,神经突起较长且相互连接成网。未见GFAP和NF200双标阳性细胞,其中,NF200阳性细胞占89.1%,GFAP阳性细胞占6.7%。提取的CNS髓鞘质经Western检测出含有Nogo-A,MAG,CSPG抑制蛋白,在浓度为200μg/ml时对培养神经元轴突生长有显著抑制效应而无明显的细胞毒性。
3、神经元细胞E3B1基因的表达变化检测
在正常培养的神经元细胞内,检测到少量E3B1mRNA和蛋白的表达,蛋白的表达主要位于神经元胞体和突起部,在轴突生长抑制时?Ш-tubulin表达明显降低,而E3B1mRNA和蛋白的表达显著升高。
4、E3B1基因的表达调控对培养神经元细胞轴突再生的影响
1)构建了针对E3B1的RNA干扰真核表达载体Abi11,Abi12,Abi13,HK和GAPDH,经抗性筛选、酶切、测序鉴定,证明重组RNAi质粒的构建符合设计要求,序列完全正确。
2)各组质粒的神经元转染率分别为34.2%(HK)、36.3%(Abi11)、33.5%(Abi12)和35.7%(Abi13)无明显差异,对照组HK对神经元E3B1mRNA和蛋白表达无影响,实验组Abi11,Abi12,Abi13各组均能使神经元E3B1mRNA和蛋白表达下调,其中抑制效果Abi13最为显著,可使神经原细胞E3B1蛋白的表达下调约61.26%。
3)转染Abi13-siRNA的神经元细胞与轴突生长抑制物共培养,神经元轴突仍能较好地生长,其中?-Ш微管蛋白也有较高水平的表达,与对照组相比差异显著。
结论
1、动态检测了脊髓损伤前后E3B1基因的表达变化,脊髓损伤后E3B1基因持续较长时间的表达增高与脊髓损伤急性期的变化密切相关,提示其可能在脊髓损伤、修复中起重要作用。也为更进一步的离体实验研究E3B1与神经元轴突再生抑制间的关系提供了依据。
2、成功分离、培养、鉴定了大鼠皮层神经元和提取、鉴定了CNS髓鞘质,确定了浓度为200μg/ml的CNS髓鞘质为实验的最适浓度,为进一步研究E3B1基因的表达变化与神经元轴突再生抑制间的关系奠定了实验基础。
3、神经元轴突生长抑制时E3B1mRNA和蛋白的表达显著升高。提示E3B1基因参与了神经元细胞轴突的生长调控,其过度表达与轴突生长抑制密切相关。
4、成功构建了针对E3B1的RNA干扰质粒载体,转染神经元能可靠地使神经元E3B1mRNA和蛋白表达下调,把对E3B1有最佳抑制效应的siRNA- Abi13转染神经元细胞,与轴突生长抑制物共培养,结果显示神经元细胞在CNS myelin存在的情况下轴突仍能较好地生长,与对照组相比差异显著,说明E3B1基因的过表达是神经元轴突生长抑制的重要因素,达到了通过调控E3B1基因表达促进神经元轴突生长的预期效果。
Background
The CNS injury is an injury with severe consequence and a great burden to the patients and their families as well as the society due to its frequent occurrence in prime of life and poor effectiveness in treatment. Therefore how to improve the repair and regeneration and reconstruct function after CNS injury is one of the hot studies in orthopedics.
The existance of axon growth inhibitors like myelin leads to the retraction and collapse of neuron axon and inhabit the regeneration of axon and repair after CNS injury.The mechamism of axon regenertion failure caused by axon growth inhibitors eventually lies in the neuronal cytoskeleton actin-depolymerization, stress fiber formation and inhabition in the cytoskeleton reconstrction. Previous studies have proved that to stimulate the neuronal cytoskeleton actin-depolymerization and reconstruct the cytoskeleton could enhance the axon growth,thus,to find a way of stimulating the neuronal cytoskeleton actin-depolymerization and reconstructing the cytoskeleton so as to release the inhibition is the key to CNS injury repair. The present studies believe that the neuronal cytoskeleton actin-depolymerization and reconstruction of the microfilament, microtubule of cytoskeleton could disinhibit axon regenertion due to the inhibitors and help the oriented axon growth in a prossess of multi-phases and multi-genes,while the existance of some essencial genes could not identified. It is the existance of such genes that make the CNS injury repair feasible for the their function of releasing of inhibitiin in the axon regeneration and enhancing the oriented growth.
E3B1(also called Abi1)was separated and identified in 1995 as a binding-protein regulating the cells growth and proliferating the Abl Non-Receptor Tyrosine Kinase(also called Eps8).The homologous protein family mainly includes Abi2、Agbp and NESH. The further study showed that the E3B1 plays most important part in the cytoskeleton reconstrction and the neurite outgrowth and its over-expression could result in the actin-depolymerization and the inhabition in the cytoskeleton reconstrction, thus E3B1 could be a critical gene regulating actin-depolymerization and cytoskeleton reconstrction. Therefore in this study, we explored the changes of E3B1 protein expression prior to and after the neurite growth inhibition by using bio-chemical technique and finished the designing, constructing, screening and transforming siRNA to inhibit the E3B1 protein expression in the neurons and enhance the regeneration of axon by using the RNA interference technology, which provided a novel idea of treating the inhibition of the anox regeneration and a new way of regenearation and repair after the CNS injury.
Methods and Materials
1. The study on the changes of E3B1 expression in the acute spinal cord injury tissue of rats.
A model of acute spinal cord injury tissue of rats was set up and the changes of E3B1 protein and E3B1mRNA in the tissues before and after spinal cord injury were observed in a dynamic way and its significance of the changes of E3B1 protein in the acute spinal cord injury was analyzed by way of immunohistochemistry, RT-PCR、Western blot.
2. The separation, culture, and identification of the rat cortex neurons & the extraction and identification of CNS myelolysis
The primary isolation and culture of the rat cortex tissue were made and its growth and morphology was observed. The quantity and shape of the neutrons were labeled by with fluorescence dye of GFAP and NF200. The CNS myelolysis was extracted and its effect on the axonal growth in different concentration was observed. Then the most effective concentration was used to make component identification by way of Western blot.
3. The identification of changes of the neuronal cells E3B1 gene expression
1) The expression distribution of the E3B1 in the neuronal cells culture was observed by means of immunohistochemistry. The E3B1 protein and E3B1mRNA expression in the neuronal cells culture was identified by using RT-PCR、Western blot.
2) The axon growth inhibitor, inhiCNS myelolysis extracted, was added into the neuronal cells culture and the E3B1 protein and E3B1mRNA expression during the inhibition of neuronal axon by using RT-PCR、Western blot.
3) The changes of E3B1 and ?Ш-tubulin expression in the neuronal cells were observed by comparing the fluorescence labeling before and after the CNS myelolysis was added. The correlation between the changes of the E3B1 gene expression and inhibition of axon growth.
4. The effect of regulation of E3B1 genes expression on the regeneration of axon growth. in the neuronal cells culture.
1) The construction of the siRNA expressing plasmid targeting the E3B1: three RNAi target sites (named as Abi11,Abi12,Abi13) targeting the E3B1(NCBI:NM-024397)a identified target sites (negative control, named as HK) and positive control GAPDH-A were selected. The pGenesil-1 eukaryotic expression vectors with the neoR mark amd GFP green fluorescent mark were selected to construct E3B1 RNAi plasmid. The resistance screening, restriction enzyme and sequencing were made.
2) The study on the efect of RNA interference on the E3B1 expression: the neuronal cells were transfected by the RNAi plasmid constructed by way of Lipofectamine 2000. The effects of RNAi targeting various sites on E3B1 genes expression were evaluated by testing the transfection efficiency with fluorescence microscope, RT-PCR and Western blot. The most effective siRNA in inhibiting E3B1 genes were screened.
3) The effect of E3B1 regulation on the axon growth: The most effective siRNA in inhibiting E3B1 genes screened were used to transfect the neuronal cells. After the resistance screening was made and the inhibitor of axon growth was added, the axon growth inhibited by E3B1 genes expression was observed and compared by use of the ?Ш-tubulin immunofluorescence and Western blot.
Results
1. The study on the changes of E3B1 genes in the acute spinal cord injury tissue of rats.
There only was little E3B1 mRNA and protein expression in normal spinal cord tissue that mainly lies in spinal cord matter. The E3B1 mRNA and protein expression increased significantly 6 hours after of the acute spinal cord injury and reach the peak at 24-48 hours after injury, then decreased gradually.
2. The separation, culture, and identification of the rat cortex neurons & the extraction and identification of CNS myelolysis
There were cells adhering 3 hours after inoculation and becoming stable in quantity and shape at 7-10 hours. The outgrowth of neuritis became longer and net. No positive cells with both GFA and NF200 marks, while the positive cells with NF200 mark were 89.1% , the positive cells with GFAP 6.7% and the rest were 4.2% which were the other types of cells with neither GFA and NF200 mark. The Nogo-A,MAG,CSPG inhibiting protein was identified in the CNS myelolysis by method of Western which had significant inhibiting effect on the axon growth at concentration of 200μg/ml without obvious cytotoxicity.
3. The identification of changes of the neuronal cells E3B1 gene expression
There was little E3B1 mRNA and protein expression in normal neuronal cells culture hat mainly lies in the neuronal somas and the neurite. The ?Ш-tubulin expression decreased significantly while the E3B1mRNA and protein expression increased when the axon growth was inhibited.
4. The effect of regulation of E3B1 genes expression on the regeneration of axon growth in the neuronal cells culture.
1) The siRNA expressing plasmids targeting the E3B1: Abi11,Abi12,Abi13,HK and GAPDH were constructed. The construction of regrouping RNAi plasmid was proved conforming to the design and the sequence after resistance screening, restriction enzyme and sequencing
2) The transfecting rates of neurons in each plasmid were 34.2%(HK)、36.3%(Abi11)、33.5%(Abi12)和35.7%(Abi13)respectively and no significant difference existing. There was no effect of HK on the E3B1mRNA and protein expression in control group while the E3B1mRNA and protein expression decreased and showed a significant inhibition effect in the experimental groups of Abi11, Abi12, Abi13. The decrease of E3B1mRNA and protein expression reached about 61.26%.
3) The co- culture of siRNA- Abi13 transfecting neurons with most effective inhibiting effect on E3B1 with the axon inhibitors showed a well growth of the neurons. The ?-Шmicrotubule showed a high expression and a significant difference was showed comparing with the control group.
Conclusion
1. The changes of E3B1 gene expression before and after spinal cord injury were identified in a dynamic way. The close correlation between the increase of E3B1 gene expression for a long period time after spinal cord injury and the change of acute spinal cord injury stage indicates that it plays critical part in the spinal cord injury and repair, which has provided an evidence for in vitro experiment in correlation between the E3B1 and axon regeneration.
2. The separation, culture, and identification of the rat cortex neurons were made and the extraction and identification of CNS myelolysis were performed successfully. It is identified that 200μg/ml of CNS cortex neuron is most effective concentration in experiment that has provided a base for experiment for correlation between the E3B1 and axon regeneration.
3. The E3B1mRNA and protein expression increased significantly when the axon growth was inhibited, indicating that the E3B1 had take part in regulating the axon growth and there existing a close correlation between the its over expression and growth inhibition, which has given a solid foundation for further regulating E3B1 expression and axon growth.
4. The siRNA expressing plasmid targeting the E3B1 was constructed successfully. The transfected neurons can surely deceased E3B1mRNA and protein expression. The co-culture of siRNA- Abi13 transfecting neurons with most effective inhibiting effect on E3B1 with the axon inhibitors showed a well growth of the neurons at the presence of CNS myelin and a significant difference with that in the control group, indicating that the over-expression of E3B1 gene is the critical factor in inhibiting the axon growth and can achieve the expected effects on enhancing the axon growth by way of E3B1 gene expression regulation.
CNS损伤是一种后果极为严重的创伤,由于疗效差且多发生于青壮年,对个人、家庭和社会都会带来巨大负担。如何促进CNS损伤后的再生修复及功能重建已成为当今骨外科学研究的热点和难点课题之一。
CNS损伤后,髓磷脂(myelin)等轴突生长抑制物的存在使神经元轴突回缩和崩溃,抑制了轴突的再生,也限制了CNS损伤后的修复。轴突生长抑制因子使轴突再生失败的作用机制,最终是通过使神经元细胞骨架肌动蛋白发生解聚、stress fiber形成、抑制细胞骨架重建而实现的。有研究证实启动神经元内肌动蛋白发生聚合,细胞骨架重建,可以促进轴突生长。因此,如何启动神经元内肌动蛋白发生聚合作用、促进神经元细胞骨架重建是解除抑制因子对轴突再生抑制、促进轴突定向生长、进而促进CNS损伤修复的关键所在。目前一般认为启动神经元内肌动蛋白发生聚合,促进微丝、微管细胞骨架重建,从而解除抑制因子对轴突再生的抑制,促进轴突定向生长,是一个多环节的、多种基因参与的过程。但在这一过程中可能存在着某个或某些关键性基因,从这些关键性基因着手,就会为解除抑制因子对轴突再生的抑制作用,促进轴突定向生长、进而促进CNS损伤修复治疗提供新的思路。
E3B1(也称为Abi1)于1995年作为调节细胞生长和增殖相关的非受体蛋白酪氨酸激酶Abl(也称为Eps8)的结合蛋白得到分离和鉴定[12]。其同源蛋白家族主要包括Abi2、Agbpl和NESH。进一步研究证实: E3B1在细胞骨架重建和细胞突起形成中发挥关键性作用,其过度表达能够引起肌动蛋白的解聚以及抑制细胞骨架重建。因此, E3B1可能是调控肌动蛋白聚合和细胞骨架重建的关键性基因。为此,本课题采用分子生物学技术,探讨神经元轴突抑制前后E3B1的表达变化规律,并通过RNA干扰(RNAi)技术,设计、构建、筛选和转染siRNA,抑制神经元内E3B1的表达,以促进轴突的再生。为轴突再生抑制的治疗提供新的思路,并为临床上促进CNS损伤后的再生修复探索新的途径。
研究内容及方法
1、E3B1基因在急性脊髓损伤大鼠脊髓组织中的表达变化研究建立大鼠急性脊髓损伤模型,采用免疫组织化学、RT-PCR、Western blot研究方法,动态观测大鼠脊髓损伤前后脊髓组织中E3B1蛋白和E3B1mRNA的表达变化,分析了E3B1基因表达变化在脊髓损伤中的意义。
2、大鼠皮层神经元的分离、培养、鉴定和CNS髓鞘质的提取、鉴定对大鼠大脑皮层组织进行原代分离培养,观察其生长和形态结构,并采用GFAP与NF200荧光标记,确认神经元的形态和数量。提取了CNS髓鞘质,观测不同浓度CNS髓鞘质对神经元轴突生长的影响,选取最适实验浓度,并采用Western blot进行成分鉴定。
3、神经元细胞E3B1基因的表达变化检测
1)采用免疫细胞化学观测了E3B1在培养神经元细胞中的表达分布,采用RT-PCR、Western blot检测培养神经元细胞中E3B1mRNA和E3B1蛋白的表达。
2)培养神经元细胞中加入提取的轴突生长抑制物CNS髓鞘质,采用RT-PCR、Western blot检测了神经元轴突抑制时E3B1蛋白和E3B1mRNA的表达变化。
3)采用免疫荧光标记对比观测了培养神经元细胞加入CNS髓鞘质前后, E3B1和?Ш-tubulin在神经元细胞中的表达变化,分析了E3B1基因表达变化与神经元轴突生长抑制的相关性。
4、E3B1基因的表达调控对培养神经元细胞轴突再生的影响
1)针对E3B1的RNA干扰质粒载体的构建:选择针对E3B1(NCBI:NM-024397)的RNAi靶位点三个(分别命名为Abi11,Abi12,Abi13)和已明确的不针对任何mRNA的RNAi靶位点一个(阴性对照,命名HK),以及阳性对照GAPDH-A,选用带有neoR选择标志和GFP绿色荧光标志的真核表达载体pGenesil-1构建E3B1RNAi质粒。并进行了抗性筛选、酶切、测序鉴定。
2) RNA干扰对神经元细胞E3B1表达的影响研究:将构建好的RNAi质粒载体通过Lipofectamine 2000依次转染培养的神经元细胞,采用荧光显微镜检测转染率,RT-PCR和Western blot检测针对E3B1不同靶位点的RNAi对神经元细胞E3B1基因表达的影响,并筛选出了对E3B1基因具有最佳抑制效应的siRNA。
3)调控E3B1表达对神经元轴突生长的影响:将筛选出的对E3B1基因具有最佳抑制效应的siRNA转染神经元细胞,经抗性筛选,加入轴突生长抑制物,采用?Ш-tubulin免疫荧光标记和Western blot对比观测E3B1基因表达抑制的神经元轴突的生长状况。
结果
1、E3B1基因在急性脊髓损伤大鼠脊髓组织中的表达变化研究
正常脊髓组织中存在少量E3B1 mRNA和蛋白表达,主要位于脊髓灰质区,急性脊髓损伤后6h E3B1 mRNA和蛋白的表达已明显升高,并于24-48h达高峰,之后逐渐降低。
2、大鼠皮层神经元的分离、培养、鉴定和CNS髓鞘质的提取、鉴定
神经元接种后约3h逐步有细胞开始贴壁, 7～10 d左右细胞形态和数量较为稳定,神经突起较长且相互连接成网。未见GFAP和NF200双标阳性细胞,其中,NF200阳性细胞占89.1%,GFAP阳性细胞占6.7%。提取的CNS髓鞘质经Western检测出含有Nogo-A,MAG,CSPG抑制蛋白,在浓度为200μg/ml时对培养神经元轴突生长有显著抑制效应而无明显的细胞毒性。
3、神经元细胞E3B1基因的表达变化检测
在正常培养的神经元细胞内,检测到少量E3B1mRNA和蛋白的表达,蛋白的表达主要位于神经元胞体和突起部,在轴突生长抑制时?Ш-tubulin表达明显降低,而E3B1mRNA和蛋白的表达显著升高。
4、E3B1基因的表达调控对培养神经元细胞轴突再生的影响
1)构建了针对E3B1的RNA干扰真核表达载体Abi11,Abi12,Abi13,HK和GAPDH,经抗性筛选、酶切、测序鉴定,证明重组RNAi质粒的构建符合设计要求,序列完全正确。
2)各组质粒的神经元转染率分别为34.2%(HK)、36.3%(Abi11)、33.5%(Abi12)和35.7%(Abi13)无明显差异,对照组HK对神经元E3B1mRNA和蛋白表达无影响,实验组Abi11,Abi12,Abi13各组均能使神经元E3B1mRNA和蛋白表达下调,其中抑制效果Abi13最为显著,可使神经原细胞E3B1蛋白的表达下调约61.26%。
3)转染Abi13-siRNA的神经元细胞与轴突生长抑制物共培养,神经元轴突仍能较好地生长,其中?-Ш微管蛋白也有较高水平的表达,与对照组相比差异显著。
结论
1、动态检测了脊髓损伤前后E3B1基因的表达变化,脊髓损伤后E3B1基因持续较长时间的表达增高与脊髓损伤急性期的变化密切相关,提示其可能在脊髓损伤、修复中起重要作用。也为更进一步的离体实验研究E3B1与神经元轴突再生抑制间的关系提供了依据。
2、成功分离、培养、鉴定了大鼠皮层神经元和提取、鉴定了CNS髓鞘质,确定了浓度为200μg/ml的CNS髓鞘质为实验的最适浓度,为进一步研究E3B1基因的表达变化与神经元轴突再生抑制间的关系奠定了实验基础。
3、神经元轴突生长抑制时E3B1mRNA和蛋白的表达显著升高。提示E3B1基因参与了神经元细胞轴突的生长调控,其过度表达与轴突生长抑制密切相关。
4、成功构建了针对E3B1的RNA干扰质粒载体,转染神经元能可靠地使神经元E3B1mRNA和蛋白表达下调,把对E3B1有最佳抑制效应的siRNA- Abi13转染神经元细胞,与轴突生长抑制物共培养,结果显示神经元细胞在CNS myelin存在的情况下轴突仍能较好地生长,与对照组相比差异显著,说明E3B1基因的过表达是神经元轴突生长抑制的重要因素,达到了通过调控E3B1基因表达促进神经元轴突生长的预期效果。
Background
The CNS injury is an injury with severe consequence and a great burden to the patients and their families as well as the society due to its frequent occurrence in prime of life and poor effectiveness in treatment. Therefore how to improve the repair and regeneration and reconstruct function after CNS injury is one of the hot studies in orthopedics.
The existance of axon growth inhibitors like myelin leads to the retraction and collapse of neuron axon and inhabit the regeneration of axon and repair after CNS injury.The mechamism of axon regenertion failure caused by axon growth inhibitors eventually lies in the neuronal cytoskeleton actin-depolymerization, stress fiber formation and inhabition in the cytoskeleton reconstrction. Previous studies have proved that to stimulate the neuronal cytoskeleton actin-depolymerization and reconstruct the cytoskeleton could enhance the axon growth,thus,to find a way of stimulating the neuronal cytoskeleton actin-depolymerization and reconstructing the cytoskeleton so as to release the inhibition is the key to CNS injury repair. The present studies believe that the neuronal cytoskeleton actin-depolymerization and reconstruction of the microfilament, microtubule of cytoskeleton could disinhibit axon regenertion due to the inhibitors and help the oriented axon growth in a prossess of multi-phases and multi-genes,while the existance of some essencial genes could not identified. It is the existance of such genes that make the CNS injury repair feasible for the their function of releasing of inhibitiin in the axon regeneration and enhancing the oriented growth.
E3B1(also called Abi1)was separated and identified in 1995 as a binding-protein regulating the cells growth and proliferating the Abl Non-Receptor Tyrosine Kinase(also called Eps8).The homologous protein family mainly includes Abi2、Agbp and NESH. The further study showed that the E3B1 plays most important part in the cytoskeleton reconstrction and the neurite outgrowth and its over-expression could result in the actin-depolymerization and the inhabition in the cytoskeleton reconstrction, thus E3B1 could be a critical gene regulating actin-depolymerization and cytoskeleton reconstrction. Therefore in this study, we explored the changes of E3B1 protein expression prior to and after the neurite growth inhibition by using bio-chemical technique and finished the designing, constructing, screening and transforming siRNA to inhibit the E3B1 protein expression in the neurons and enhance the regeneration of axon by using the RNA interference technology, which provided a novel idea of treating the inhibition of the anox regeneration and a new way of regenearation and repair after the CNS injury.
Methods and Materials
1. The study on the changes of E3B1 expression in the acute spinal cord injury tissue of rats.
A model of acute spinal cord injury tissue of rats was set up and the changes of E3B1 protein and E3B1mRNA in the tissues before and after spinal cord injury were observed in a dynamic way and its significance of the changes of E3B1 protein in the acute spinal cord injury was analyzed by way of immunohistochemistry, RT-PCR、Western blot.
2. The separation, culture, and identification of the rat cortex neurons & the extraction and identification of CNS myelolysis
The primary isolation and culture of the rat cortex tissue were made and its growth and morphology was observed. The quantity and shape of the neutrons were labeled by with fluorescence dye of GFAP and NF200. The CNS myelolysis was extracted and its effect on the axonal growth in different concentration was observed. Then the most effective concentration was used to make component identification by way of Western blot.
3. The identification of changes of the neuronal cells E3B1 gene expression
1) The expression distribution of the E3B1 in the neuronal cells culture was observed by means of immunohistochemistry. The E3B1 protein and E3B1mRNA expression in the neuronal cells culture was identified by using RT-PCR、Western blot.
2) The axon growth inhibitor, inhiCNS myelolysis extracted, was added into the neuronal cells culture and the E3B1 protein and E3B1mRNA expression during the inhibition of neuronal axon by using RT-PCR、Western blot.
3) The changes of E3B1 and ?Ш-tubulin expression in the neuronal cells were observed by comparing the fluorescence labeling before and after the CNS myelolysis was added. The correlation between the changes of the E3B1 gene expression and inhibition of axon growth.
4. The effect of regulation of E3B1 genes expression on the regeneration of axon growth. in the neuronal cells culture.
1) The construction of the siRNA expressing plasmid targeting the E3B1: three RNAi target sites (named as Abi11,Abi12,Abi13) targeting the E3B1(NCBI:NM-024397)a identified target sites (negative control, named as HK) and positive control GAPDH-A were selected. The pGenesil-1 eukaryotic expression vectors with the neoR mark amd GFP green fluorescent mark were selected to construct E3B1 RNAi plasmid. The resistance screening, restriction enzyme and sequencing were made.
2) The study on the efect of RNA interference on the E3B1 expression: the neuronal cells were transfected by the RNAi plasmid constructed by way of Lipofectamine 2000. The effects of RNAi targeting various sites on E3B1 genes expression were evaluated by testing the transfection efficiency with fluorescence microscope, RT-PCR and Western blot. The most effective siRNA in inhibiting E3B1 genes were screened.
3) The effect of E3B1 regulation on the axon growth: The most effective siRNA in inhibiting E3B1 genes screened were used to transfect the neuronal cells. After the resistance screening was made and the inhibitor of axon growth was added, the axon growth inhibited by E3B1 genes expression was observed and compared by use of the ?Ш-tubulin immunofluorescence and Western blot.
Results
1. The study on the changes of E3B1 genes in the acute spinal cord injury tissue of rats.
There only was little E3B1 mRNA and protein expression in normal spinal cord tissue that mainly lies in spinal cord matter. The E3B1 mRNA and protein expression increased significantly 6 hours after of the acute spinal cord injury and reach the peak at 24-48 hours after injury, then decreased gradually.
2. The separation, culture, and identification of the rat cortex neurons & the extraction and identification of CNS myelolysis
There were cells adhering 3 hours after inoculation and becoming stable in quantity and shape at 7-10 hours. The outgrowth of neuritis became longer and net. No positive cells with both GFA and NF200 marks, while the positive cells with NF200 mark were 89.1% , the positive cells with GFAP 6.7% and the rest were 4.2% which were the other types of cells with neither GFA and NF200 mark. The Nogo-A,MAG,CSPG inhibiting protein was identified in the CNS myelolysis by method of Western which had significant inhibiting effect on the axon growth at concentration of 200μg/ml without obvious cytotoxicity.
3. The identification of changes of the neuronal cells E3B1 gene expression
There was little E3B1 mRNA and protein expression in normal neuronal cells culture hat mainly lies in the neuronal somas and the neurite. The ?Ш-tubulin expression decreased significantly while the E3B1mRNA and protein expression increased when the axon growth was inhibited.
4. The effect of regulation of E3B1 genes expression on the regeneration of axon growth in the neuronal cells culture.
1) The siRNA expressing plasmids targeting the E3B1: Abi11,Abi12,Abi13,HK and GAPDH were constructed. The construction of regrouping RNAi plasmid was proved conforming to the design and the sequence after resistance screening, restriction enzyme and sequencing
2) The transfecting rates of neurons in each plasmid were 34.2%(HK)、36.3%(Abi11)、33.5%(Abi12)和35.7%(Abi13)respectively and no significant difference existing. There was no effect of HK on the E3B1mRNA and protein expression in control group while the E3B1mRNA and protein expression decreased and showed a significant inhibition effect in the experimental groups of Abi11, Abi12, Abi13. The decrease of E3B1mRNA and protein expression reached about 61.26%.
3) The co- culture of siRNA- Abi13 transfecting neurons with most effective inhibiting effect on E3B1 with the axon inhibitors showed a well growth of the neurons. The ?-Шmicrotubule showed a high expression and a significant difference was showed comparing with the control group.
Conclusion
1. The changes of E3B1 gene expression before and after spinal cord injury were identified in a dynamic way. The close correlation between the increase of E3B1 gene expression for a long period time after spinal cord injury and the change of acute spinal cord injury stage indicates that it plays critical part in the spinal cord injury and repair, which has provided an evidence for in vitro experiment in correlation between the E3B1 and axon regeneration.
2. The separation, culture, and identification of the rat cortex neurons were made and the extraction and identification of CNS myelolysis were performed successfully. It is identified that 200μg/ml of CNS cortex neuron is most effective concentration in experiment that has provided a base for experiment for correlation between the E3B1 and axon regeneration.
3. The E3B1mRNA and protein expression increased significantly when the axon growth was inhibited, indicating that the E3B1 had take part in regulating the axon growth and there existing a close correlation between the its over expression and growth inhibition, which has given a solid foundation for further regulating E3B1 expression and axon growth.
4. The siRNA expressing plasmid targeting the E3B1 was constructed successfully. The transfected neurons can surely deceased E3B1mRNA and protein expression. The co-culture of siRNA- Abi13 transfecting neurons with most effective inhibiting effect on E3B1 with the axon inhibitors showed a well growth of the neurons at the presence of CNS myelin and a significant difference with that in the control group, indicating that the over-expression of E3B1 gene is the critical factor in inhibiting the axon growth and can achieve the expected effects on enhancing the axon growth by way of E3B1 gene expression regulation.
引文
1. Ng CE, Tang BL.Nogos and the Nogo-66 receptor: factors inhibiting CNS neuron regeneration. J Neurosci Res 2002;67(5):559-565
2. Liu BP, Fournier A, GrandPre T, et al. Myelin-Associated Glycoprotein as a Functional Ligand for the Nogo-66 Receptor. Science 2002;297(5584): 1190-1193.
3. Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature 2002;417(6892):941-944
4. Wang KC, Kim JA, Sivasankaran R, et al. p75 interacts with the Nogo receptor as a co-receptor for Nogo,MAG and OMgp. Nature 2002;420(6911):74-78.
5. Gentry JJ, Barker PA,Carter BD. The p75 neurotrophin receptor: multiple interactors and numerous functions. Prog Brain Res. 2004;146:25-39
6. Alan Hall. Rho GTPases and the Actin Cytoskeleton. Science 1998;279:509-514
7. Barbara Niederost, Thomas Oertle, Jens Fritsche, et al. Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Rac1.Neurosci 2002,22(23):10368-10376
8. Masoud Shekarabi, Timothy E. K. The Netrin-1 Receptor DCC Promotes Filopodia Formation and Cell Spreading by Activating Cdc42 and Rac1.Mol Cell Neurosci 2002;19:1-17
9. Jaimie F. Borisoff, Carmen C.M. Chan,Gordon W. Hiebert, et al. Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates. Mol Cell Neurosci 2003;22:405-416
10. Paul A. Janmey. Creating a niche in the cytoskeleton: Actin reorganization by a protein kinase. PNAS 2001;98(26):14745-14747
11. Dickson B.j. Rho in growth cone guidance. Curr Opin Neurobio 2001;11:103-110
12. Shi Y. Alin K. Goff S. P. Abl-interactor-1, a novel SH3 protein binding to the carboxy-terminal portion of the Abl protein, suppresses v-abl transforming activity.Genes Dev 1995;9(21):2583-2597
13. Giorgio Scita, Pierluigi Tenca, Liliana B. Areces, et al. An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.JCB2001;154(5):1031-1044
14. Innocenti M, Frittoli E, Ponzanelli I,et al. Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abil, and Sos-1.JCB 2003;160(1)17-23
15. Steffen A, Rottner K,Ehinger J, et al. Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation. EMBO J 2004;23(4):749-759
16. Patricia Kunda, Gavin Craig,Veronica Dominguez,et al. Abi,Sral, and Kette control the stability and location of SCAR/WAVE to regulate the formation of actin-baseded protrusions. Curr Biol2003;13:1867-1875
17. Fan PD, Goff SP. Abl interactor 1 binds to sos and inhibits epidermal growth factor and v-Abl-induced activation of extracellular signal-regulated kinases. Mol Cell Biol 2000;20(20):7591-7601
18. Courtney KD, Grove M, Vandongen H, Vandongen A, et al. Localization and phosporylation of Abl-interactor proteins, Abi1 and Abi2, in the developing nervous system. Mol Cell Neurosci 2000;16(3):244-257
19. Stradal T, Courtney KD, Rottner K, et al. The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia. Curr Biol 2001; 11(11):891-895
1. Gennadij Raivich and Milan Makwana .The making of successful axonal regeneration: Genes, molecules and signal transduction pathways Brain Research Reviews, Volume 53, Issue 2, February 2007, 287-311
2. Kuo-Cheng Huang, Zivart Yasruel, Claude Guérin ,et al.Interaction of the Coxsackie and adenovirus receptor (CAR) with the cytoskeleton: Binding to actinFEBS Letters, Volume 581, Issue 14, 12 June 2007, 2702-2708
3. Pramod K. Allani, Tak Sum, et al.A comparative study of the effect of oxidative stress on the cytoskeleton in human cortical neurons Toxicology and Applied Pharmacology, Volume 196, Issue 1, 1 April 2004, 29-36
4. Ching-Hui Huang, Ju-Chien Cheng, Jin-Chung,et al.Evaluation of the role of Disabled-2 in nerve growth factor-mediated neurite outgrowth and cellular signaling Cellular Signalling, Volume 19, Issue 6, June 2007, 1339-1347
5. Rachel de las Heras, Iris Depaz, Vincent Jaquet ,et al.Neuronal protein 22 colocalises with both the microtubule and microfilament cytoskeleton in neurite-like processes Brain Research, Volume 1128, 12 January 2007, 12-20
6. Bettina Büttner, Christoph Kannicht, Werner Reutter,et al.The neural cell adhesion molecule is associated with major components of the cytoskeleton Biochemical and Biophysical Research Communications, Volume 310, Issue 3, 24 October 2003, 967-971
7. E. Nyatia ,D.M. Lang Localisation and expression of a myelin associated neurite inhibitor, Nogo-A and its receptor Nogo-receptor by mammalian CNS cells Research in Veterinary Science, Volume 83, Issue 3, December 2007, 287-301
8. Oliver Weinmann, Lisa Schnell, Arko Ghosh,et al.Intrathecally infused antibodies against Nogo-A penetrate the CNS and downregulate the endogenous neurite growth inhibitor Nogo-A.Molecular and Cellular Neuroscience, Volume 32, Issues 1-2, May-June 2006, 161-173
9. Rüdiger Schweigreiter, Adrian R.,Walmsley,et al.Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA.Molecular and Cellular Neuroscience, Volume 27, Issue 2, 1 October 2004, 163-174
10. Ferdinando Rossi, Sara Gianola and Luigi Corvetti.Regulation of intrinsic neuronal properties for axon growth and regeneration.Progress in Neurobiology, Volume 81, Issue 1, January 2007, 1-28
11. H. Tanaka, T. Yamashita, K. Yachi,et al.Cytoplasmic p21Cip1/WAF1 enhances axonal regeneration and functional recovery after spinal cord injury in rats. Neurosc- ience, Volume 127, Issue 1, 2004, 155-164
12. Marco Domeniconi and Marie T. Filbin .Overcoming inhibitors in myelin to promote axonal regeneration.Journal of the Neurological Sciences, Volume 233, Issues 1-2, 15 June 2005, 43-47
13. Jennifer J. Gentry, Philip A,et al.The p75 neurotrophin receptor: multiple interactors and numerous functions.Progress in Brain Research, Volume 146, 2004, Pages 25-39
14. Altman J. Programmed cell death: the paths to suicide. Trends Neurosci, 1992, 15(8): 278-280.
15. Patrick Vourc'h and Christian Andres.Oligodendrocyte myelin glycoprotein (OMgp): evolution, structure and function.Brain Research Reviews, Volume 45, Issue 2, May 2004, 115-124
16. G.K.T. Chu, W. Yu,M.G. Fehlings.The p75 neurotrophin receptor is essential for neuronal cell survival and improvement of functional recovery after spinal cord injury.Neuroscience, Volume 148, Issue 3, 7 September 2007, Pages 668-682
17. Marco Domeniconi, NiccolòZampieri, Tim Spencer,et al.MAG Induces Regulated Intramembrane Proteolysis of the p75 Neurotrophin Receptor to Inhibit Neurite Outgrowth.Neuron, Volume 46, Issue 6, 16 June 2005, 849-855
18. Lixin Fan, Saulius Girnius,Bruce Oakley,et al.Support of trigeminal sensory neurons bynonneuronal p75 neurotrophin receptors.Developmental Brain Research, Volume 150, Issue 1, 19 May 2004, Pages 23-39
19. Yuki Miyamoto, Junji Yamauchi, Atsushi Sanbe,et al. Dock6, a Dock-C subfamily guanine nucleotide exchanger, has the dual specificity for Rac1 and Cdc42 and regulates neurite outgrowth.Experimental Cell Research, Volume 313, Issue 4, 15 February 2007, Pages 791-804
20. Charles E. Laurent and Thomas E. Smithgall.The c-Fes tyrosine kinase cooperates with the breakpoint cluster region protein (Bcr) to induce neurite extension in a Rac- and Cdc42-dependent manner.Experimental Cell Research, Volume 299, Issue 1, 10 September 2004, Pages 188-198
21. Denice L. Major,Susann M. Brady-Kalnay.Rho GTPases regulate PTPμ-mediated nasal neurite outgrowth and temporal repulsion of retinal ganglion cell neurons.Molecular and Cellular Neuroscience, Volume 34, Issue 3, March 2007, Pages 453-467
22. Anthony D. Couvillon and John H. Exton .Role of heterotrimeric G-proteins in lysophosphatidic acid-mediated neurite retraction by RhoA-dependent and -independent mechanisms in N1E-115 cells.Cellular Signalling, Volume 18, Issue 5, May 2006, Pages 715-728
23. Zubair Ahmed, Russell G. Dent, Ellen L ,et al.Disinhibition of neurotrophin-induced dorsal root ganglion cell neurite outgrowth on CNS myelin by siRNA-mediated knockdown of NgR, p75NTR and Rho-A.Molecular and Cellular Neuroscience, Volume 28, Issue 3, March 2005, Pages 509-523
24. Barbara Niederost, Thomas Oertle, Jens Fritsche, et al. Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Rac1.Neurosci 2002,22(23):10368-10376
25. Charles E. Laurent ,Thomas E. Smithgall The c-Fes tyrosine kinase cooperates with the breakpoint cluster region protein (Bcr) to induce neurite extension in a Rac- and Cdc42-dependent manner.Experimental Cell Research, Volume 299, Issue 1, 10 September 2004, Pages 188-198
26. Masoud Shekarabi, Timothy E. K. The Netrin-1 Receptor DCC Promotes Filopodia Formation and Cell Spreading by Activating Cdc42 and Rac1.Mol Cell Neurosci 2002;19:1-17
27. Jaimie F. Borisoff, Carmen C.M. Chan,Gordon W. Hiebert, et al. Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates. Mol Cell Neurosci 2003;22:405-416
28. Paul A. Janmey. Creating a niche in the cytoskeleton: Actin reorganization by a protein kinase. PNAS 2001;98(26):14745-14747
29. Dickson B.j. Rho in growth cone guidance. Curr Opin Neurobio 2001;11:103-110
30. Shi Y. Alin K. Goff S. P. Abl-interactor-1, a novel SH3 protein binding to the carboxy-terminal portion of the Abl protein, suppresses v-abl transforming activity.Genes Dev 1995;9(21):2583-2597
31. Giorgio Scita, Pierluigi Tenca, Liliana B. Areces, et al. An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and forthe proper localization of the Rac-based actin-polymerizing machine.JCB 2001; 154 (5):1031-1044
32. Innocenti M, Frittoli E, Ponzanelli I,et al. Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abil, and Sos-1.JCB 2003;160(1)17-23
33. Steffen A, Rottner K,Ehinger J, et al. Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation. EMBO J 2004;23(4):749-759
1. Falconer JC , Narayana PA , BhattacharieeM, et al . Characterization of an experiental spinal cord injury model suing waveformand morphometric analysis [ J ] . Spine , 1996 , 21 (1) : 104 - 112.
2. Khan T , Havey RM, Sayers ST , et al . Animal models of spinal cord contusion injuries [ J ] . Las Anim Sci , 1999 , 49 (2) : 161 - 172.
3. Hiruma S , Otsuka K, Satou T. et al . Simple and reproducible model of rat spinal cord injury induced by a controlled cortical impact device [J ] . Neurol Res , 1999 , 21 (3) :313 - 323.
4. Eugen J , Dolan , EnsorE , et al . The effect of spinal distraction on regional spinal cord blood flow in cats [J ] . J Neurosurg , 1980 , 53 (9) : 756 - 764.
5. Jarzem PF , Kostuik JP , Filiaggi M, et al . Spinal cord distraction : an vitro study of length , tension and tissue pressure [J ] . J Spine Disord , 1991 , 4 (2) : 177 - 182.
6. Maiman OJ , Myklebust JB , Ho Kc , et al . Experimental spinal cord injury produced by axial tension [J ] . J Spinal Disord , 1989 , 232 (1) : 6 - 13.
7. Allen AR,Surgery of Experimental Lesion of Spinal Cord Equivalent to Crush injury of fracture dislocation of spinal column.JAMA,1911,57:878-880.
8. Nystrom B , Berglund JE , Bergquist E. Methodological analysis of an experimentalspinal cord compression model in the rat [ J ] . J Acta Neurol Scand , 1988 , 78 (4) : 460 - 466.
9. Hashimoto T , FuKuta. New spinal cord injury model produced by spinal cord compression in the rat [ J ] . J pharmaol methods , 1990 , 23 (3) : 203 - 212.
10. Sato K, Konno S , Yabuki S , et al . Amodel for acute , chronic and delayed graded compression of the dog cauda equine [J ] . Spine , 1995 , 20 ( 22 ) : 2386 -2391.
11. Borgens RB. Functional recovery after spinal hemisection in guinea pigs the effects of applied electric field [ J ] . J Comp Neurol , 1990 , 96 (6) : 634 - 640.
12. Colak A , Nurlu G, Acikgoz B , et al . a new experimental model for further investigation of the secondary changes following spinal cord injury [ J ] . J Neurosurg Sci , 1994 , 38 (2) : 111 - 116.
13. Lo YC , Mcbrie WH , Wither HR. The effect of single doses of radiation on mouse spinal cord [ J ] . Int J Radiat Oncol Biol Phys , 1992 , 22 (1) : 57 - 63.
14. Tarlov IM, Klinger H. Spinal cord compression studies I. Experimental techniques to produce acute and gradual compression [J ] . J A M A. Arch , Neurol &Psychiat , 1953 , 70 (7) : 813 - 819.
15. Tator CH, Fehlings LG. Review of the secordary injury theory of acute spinal cord trauma with emphasis on vascular mechanism [J ]. J Newrosurg , 1991 , 75 (1) : 15 - 26.
1. Oliver Weinmann, Lisa Schnell, Arko Ghosh,et al.Intrathecally infused antibodies against Nogo-A penetrate the CNS and downregulate the endogenous neurite growth inhibitor Nogo-A.Molecular and Cellular Neuroscience, Volume 32, Issues 1-2, May-June 2006, 161-173
2. Rüdiger Schweigreiter, Adrian R.,Walmsley,et al.Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA.Molecular and Cellular Neuroscience, Volume 27, Issue 2, 1 October 2004, 163-174
3. H. Tanaka, T. Yamashita, K. Yachi,et al.Cytoplasmic p21Cip1/WAF1 enhances axonal regeneration and functional recovery after spinal cord injury in rats. Neuroscience, Volume 127, Issue 1, 2004, 155-164
4. Marco Domeniconi and Marie T. Filbin .Overcoming inhibitors in myelin to promoteaxonal regeneration.Journal of the Neurological Sciences, Volume 233, Issues 1-2, 15 June 2005, 43-47
5. Altman J. Programmed cell death: the paths to suicide. Trends Neurosci, 1992, 15(8): 278-280.
6. Patrick Vourc'h and Christian Andres.Oligodendrocyte myelin glycoprotein (OMgp): evolution, structure and function.Brain Research Reviews, Volume 45, Issue 2, May 2004, 115-124
7. Philippe P. Monnier, Ana Sierra,et al.The Rho/ROCK pathway mediates neurite growth-inhibitory activity associated with the chondroitin sulfate proteoglycans of the CNS glial scar.Molecular and Cellular Neuroscience, Volume 22, Issue 3, March 2003, 319-330
8. Diane M. Snow, Jeffrey D. Smith ,et al.Neurite elongation on chondroitin sulfate proteoglycans is characterized by axonal fasciculation.Experimental Neurology, Volume 182, Issue 2, August 2003, Pages 310-321
9. Li S,Strittmatter SM.Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury.Neurosci,2003,23(10):4219-4227.
10. Li SX,Liu BP,Budel S.Blockade of Nogo-66,myelin-associated glycoprotein,and oligodendrocyte myelin glyeoprotein by soluble Nogo-66 receptor promotes axonal sprouting and after spinal injury.Neuroscience,2004,24(46):10511-10520.
11. Ruitenberg M J,Levison DB,Lee SV,et al.Nt-3expression from engineered olfactory ensheathing glia promotes spinal sparing and regeneration.Brain, 2005,128(4): 839-853.
12. Xu G,Nie DY,Chen JT,et al.Recombinant DNA vaceine encoding multiple domains related to inhibition of neurite outgroeth:a potentialstrategy for axonal regeneration.Journal of neurochemistry.2004,91(4):1018-1023
13. Jaimie F.Borisoff,Carmen C.M.Chan,Gordon W.Hiebert,et al.Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates.Mol Cell Neurosci 2003;22:405-416
14. Patricia Kunda,Gavin Craig,Veronica Dominguez,et al.Abi,Sral,and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions.Curr Biol 2003;13:1867-1875
15. Courtney KD,Grove M,Vandongen H,Vandongen A,et al.Localization and phosphorylation of Abl-interactor proteins,Abi1 and Abi-2,in the developing nevous system.Mol Cell Neurosci 2002;16(3):244-257
16. Stradal T,Courtney KD,Rottner K,et al.The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia.Curr Biol 2002;11(11):891-895
17. Paul A.Janmey.Creating a niche in the cytoskeleton:Actin reorganization by a protein kinase.PNAS 2002;98(26):14745-14747
18. Innocenti M,Frittoli E,Ponzanelli I,et al.Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abi1,and Sos-1.JCB 2003;160(1):17-23
19. Veronika Jenei,Tommy Andersson,Judit Jakus,et al.E3B1,a human homologue of the mous gene product Abi-1,sensitizes activation of Rap1 in response to epidermal growth factor.Experimental Cell Research 2005;310(2):463-473
20. M.Innocenti,P.Tenca,E.Frittoli,I.Ponzanelli,J.R.Falck,S.M.Brachmann,P.P.Di Fiore and G.Scita,Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abi1,and sos-1,J.Cell Biol.160(2003),PP.17-23
21. Patricia Kunda,Gavin Craig,Veronica Dominguez,et al.Abi,Sral,and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions.Curr Biol 2003;13:1867-1875
22. Steffen A,Rottner K,Ehinger J,et al.Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation.EMBO J 2004;23(4):749-759
1. Kuhn TB. Growing and working with spinal motor neurons. Methods Cell Biol, 2003, 71(1): 67-97.
2. Van Damme P, Van Den BL, Van Houtte E, et al. Na+ entry through AMPA receptors results in voltage-gated K+ channel blockade in cultured rat spinal cord motoneurons. J Neurophysiol. 200, 88(2): 965-972.
3. Guigoni C, Coulon P. Rabies virus is not cytolytic for rat spinal motoneurons in vitro. J Neurovirol, 2002, 8(4): 306-317.
4. Mouveroux JM, Lakke EA, Marani E. Lumbar spinal cord explants from neonatal rat display age-related decrease of outgrowth in culture. Neurosci Lett, 2001, 311(2): 69-72.
5. Erkman L, Touzeau G, Bertrand D, et al. Characterization of dissociated monolayer cultures of human spinal cord. Brain Research Bulletin, 1989, 22: 57-65.
6. Michikawa M, Lim KT, McLarnon JG, et al. Oxygen radical induced neurotoxicity in spinal cord neuron cultures. Neurosci Res, 1994, 37: 62-70.
7. Andersona KN, Potterb AC, Piccenna LG, et al. Isolation and culture of motor neurons from the newborn mouse spinal cord. Brain Research Protocols, 2004, 12: 132– 136.
8. Vejsada R, Tseng JL, Lindsay RM, et al. Synergistic but transient rescue effects of BDNF and GDNF on axotomized neonatal motoneurons. Neuroscience, 1998, 84(1): 129-139.
9. Chen B, Hammonds-Odie L, Perron J, et al. SHP-2 Mediates Target-Regulated Axonal Termination and NGF-Dependent Neurite Growth in Sympathetic Neurons. Dev Biol, 2002, 252(2): 170-187.
10. Lindholm D, Mercer EA, Yu LY, et al. Neuronal apoptosis inhibitory protein: structural requirements for hippocalcin binding and effects on survival of NGF-dependent sympathetic neurons. BBA-Proteins Proteomics, 2002, 1600(1-2): 138-147.
11. Brian A, Pierchala T, ilbrandt J, et al. NGF Utilizes c-Ret Via a Novel GFL–Independent, Inter-RTK Signaling Mechanism to Maintain the Trophic Status of Mature Sympathetic Neurons. Neuron, 2002, 33(2): 261-273.
12. Bataille S, Portalier P, Coulon P, et al. Influence of acetylcholinesterase on embryonic spinal rat motoneurons growth in culture: a quantitative mophometric study. Eur J Neurosci, 1998, 10: 560-572.
13. Camu W and Henderson, CE. Purification of embryonic rat motoneurons by panning on a monoclonal antibody to the low affinity NGF receptor. J Neurosci Methods, 1992, 44(1): 59-70.
14. Altman J. Programmed cell death: the paths to suicide. Trends Neurosci, 1992, 15(8): 278-280.
15. Oppenheim RW. Cell death during development of the nervous system. Annu Rev Neurosci, 1991, 14: 453-501.
16. Sendtner M, Pei G, Beck M, et al. Developmental motoneuron cell death and neurotrophic factors. Cell Tissue Res, 2000, 301: 71–84.
17. Haastert K, Grosskreutz J, Jaeckel M, et al. Rat embryonic motoneurons in long-term co-culture with Schwann cells—a system to investigate motoneuron diseases on a cellular level in vitro. J Neurosci Methods, 2005, 142: 275–284.
18. Martinou IS, Desagher R, Eskes B, et al. The release of cytochrome c from mitochondria during apoptosis of NGF-deprived sympathetic neurons is a reversible event. J Cell Biol, 1999, 144(5): 883–889.
19. Brodski C, Schaubmar A, Dechant G. Opposing functions of GDNF and NGF in the development of cholinergic and noradrenergic sympathetic neurons. Mol Cel Neurosci, 2002, 19(4): 528-538.
20. Henderson CE, Phillips HS, Pollock RA, et al. GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle[J]. Science, 1994,266(5187): 1062-1064.
21. Carriedo SG, Sensi SL, Yin HZ, et al. AMPA exposure induce mitochondrial Ca2+ overload and ROS generation in spinal motor neurons in vitro. J Neurosci, 2000, 20(1): 240-250.
22. Roy J, Minotti S, Dong L, et al. Glutamate potentiates the toxicity of mutant Cu/Zn-superoxide dismutase in motor neurons by postsynaptic calcium-dependentmechanism. J Neurosci, 1998, 18(23): 9673-9684.
1. H. Tanaka, T. Yamashita, K. Yachi,et al.Cytoplasmic p21Cip1/WAF1 enhances axonal regeneration and functional recovery after spinal cord injury in rats. Neuroscience, Volume 127, Issue 1, 2004, 155-164
2. Marco Domeniconi and Marie T. Filbin .Overcoming inhibitors in myelin to promote axonal regeneration.Journal of the Neurological Sciences, Volume 233, Issues 1-2, 15 June 2005, 43-47
3. Altman J. Programmed cell death: the paths to suicide. Trends Neurosci, 1992, 15(8): 278-280.
4. Patrick Vourc'h and Christian Andres.Oligodendrocyte myelin glycoprotein (OMgp): evolution, structure and function.Brain Research Reviews, Volume 45, Issue 2, May 2004, 115-124
5. Philippe P. Monnier, Ana Sierra,et al.The Rho/ROCK pathway mediates neurite growth-inhibitory activity associated with the chondroitin sulfate proteoglycans of the CNS glial scar.Molecular and Cellular Neuroscience, Volume 22, Issue 3, March 2003, 319-330
6. Diane M. Snow, Jeffrey D. Smith ,et al.Neurite elongation on chondroitin sulfate proteoglycans is characterized by axonal fasciculation.Experimental Neurology, Volume 182, Issue 2, August 2003, Pages 310-321
7. Monnier PP , Sierra A , Schwab JM, et al. The Rho/ ROCK pathway mediates neurite growth2inhibitory activity associated with the chondroitin sulfate proteoglycans of the CNS glial scar. Mol Cell Neurosci , 2003 , 22÷319～330.
8. Emilie Pacary, Emmanuelle Tixier, Florence Coulet ,et al.Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth.Molecular and Cellular Neuroscience, Volume 35, Issue 3, July 2007, Pages 409-423
9. Philippe P. Monnier, Ana Sierra,The Rho/ROCK pathway mediates neurite growth-inhibitory activity associated with the chondroitin sulfate proteoglycans of the CNS glial scar.Molecular and Cellular Neuroscience, Volume 22, Issue 3, March 2003, Pages 319-330
10. Rüdiger Schweigreiter, Adrian R ,et al.Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA.Molecular and Cellular Neuroscience, Volume 27, Issue 2, 1 October 2004, Pages 163-174
11. Changman Zhou, Yun Li .HBO suppresses Nogo-A, Ng-R, or RhoA expression in the cerebral cortex after global ischemia.Biochemical and Biophysical Research Communications, Volume 309, Issue 2, 19 September 2003, Pages 368-376
12. Binhai Zheng, Carole Ho, Shuxin Li .Lack of Enhanced Spinal Regeneration in Nogo-Deficient MiceNeuron, Volume 38, Issue 2, 24 April 2003, Pages 213-224
13. Aaron W. McGee and Stephen M. Strittmatter .The Nogo-66 receptor: focusing myelin inhibition of axon regeneration.Trends in Neurosciences, Volume 26, Issue 4, April 2003, Pages 193-198
14. Wang KC, Kim JA, Sivasankaran R, et al. p75 interacts with the Nogo receptor as a co-receptor for Nogo,MAG and OMgp. Nature 2002;420(6911):74-78.
15. Gentry JJ, Barker PA,Carter BD. The p75 neurotrophin receptor: multiple interactors and numerous functions. Prog Brain Res. 2004;146:25-39
16. Alan Hall. Rho GTPases and the Actin Cytoskeleton. Science 1998;279:509-514
17. Zubair Ahmed,Russell G,Dent,et al.Disinhibition of neurotrophin-induced dorsal root ganglion cell neurite outgrowth on CNS nyelin by siRNA-mediated knockdown of NgR,p75NTR and Rho-A.Mol.Cell.Neurosci.2005,28,509-523.
1. Ng CE, Tang BL.Nogos and the Nogo-66 receptor: factors inhibiting CNS neuron regeneration. J Neurosci Res 2002;67(5):559-565
2. Liu BP, Fournier A, GrandPre T, et al. Myelin-Associated Glycoprotein as a Functional Ligand for the Nogo-66 Receptor. Science 2002;297(5584): 1190-1193.
3. Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature 2002;417(6892):941-944
4. Wang KC, Kim JA, Sivasankaran R, et al. p75 interacts with the Nogo receptor as a co-receptor for Nogo,MAG and OMgp. Nature 2002;420(6911):74-78.
5. Gentry JJ, Barker PA,Carter BD. The p75 neurotrophin receptor: multiple interactors and numerous functions. Prog Brain Res. 2004;146:25-39
6. Alan Hall. Rho GTPases and the Actin Cytoskeleton. Science 1998;279:509-514
7. Barbara Niederost, Thomas Oertle, Jens Fritsche, et al. Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Rac1.Neurosci 2002,22(23):10368-10376
8. Masoud Shekarabi, Timothy E. K. The Netrin-1 Receptor DCC Promotes Filopodia Formation and Cell Spreading by Activating Cdc42 and Rac1.Mol Cell Neurosci 2002;19:1-17
9. Jaimie F. Borisoff, Carmen C.M. Chan,Gordon W. Hiebert, et al. Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates. Mol Cell Neurosci 2003;22:405-416
10. Paul A. Janmey. Creating a niche in the cytoskeleton: Actin reorganization by a protein kinase. PNAS 2001;98(26):14745-14747
11. Dickson B.j. Rho in growth cone guidance. Curr Opin Neurobio 2001;11:103-110
12. Shi Y. Alin K. Goff S. P. Abl-interactor-1, a novel SH3 protein binding to the carboxy-terminal portion of the Abl protein, suppresses v-abl transforming activity.Genes Dev 1995;9(21):2583-2597
13. Giorgio Scita, Pierluigi Tenca, Liliana B. Areces, et al. An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.JCB 2001;154(5):1031-1044
14. Innocenti M, Frittoli E, Ponzanelli I,et al. Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abil, and Sos-1.JCB 2003;160(1)17-23
15. Steffen A, Rottner K,Ehinger J, et al. Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation. EMBO J 2004;23(4):749-759
16. Kazuhiro Ishiguro, Zhifang Cao, Marco Lopez Ilasca ,et al.Wave2 activates serum response element via its VCA region and functions downstream of Rac.Experimental Cell Research, Volume 301, Issue 2, 10 December 2004, Pages 331-337.
17. Patricia Kunda, Gavin Craig,Veronica Dominguez,et al. Abi,Sral, and Kette control the stability and location of SCAR/WAVE to regulate the formation of actin-baseded protrusions. Curr Biol2003;13:1867-1875
18. Fan PD, Goff SP. Abl interactor 1 binds to sos and inhibits epidermal growth factor and v-Abl-induced activation of extracellular signal-regulated kinases. Mol Cell Biol 2000;20(20):7591-7601
19. Courtney KD, Grove M, Vandongen H, Vandongen A, et al. Localization and phosporylation of Abl-interactor proteins, Abi1 and Abi2, in the developing nervous system. Mol Cell Neurosci 2000;16(3):244-257
20. Stradal T, Courtney KD, Rottner K, et al. The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia. Curr Biol 2001;11(11):891-895
1. Fire A,Xu S,Montgomery M K,et al.Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans.Nature,1998,391(6669):806-811.
2. Bernstin E,Caudy AA,Hammond SM,et al.Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature,2001,409 (6818):363-366.
3. Hannon GJ.RNA interference.Nature,2002,418(6894):244-251
4. Zeng Y,Cullen Br.RNA interference in human cells is restricted to the cytoplasm.RNA,2002,8:855-867
5. Zamore PD.RNA interference:listening to the sound of silence.Nat Struct Biol,2001,8(9):746-750
6. Zheng ZM , Tang S , Tao Mf. Development of Resistance to RNAi in Mammalian Cells[J ] . Science , 2005 ,1058 : 105 - 118.
7. Makoto Inaki, Takahiko Utsugi, Fumitoshi Onoda ,et al.A novel class of temperature-sensitive mutants generated by RNAi-mediated knockdown. Biochemical and Biophysical Research Communications, Volume 365, Issue 3, 18 January 2008, 472-477
8. Yuan Han, Wenxin Qin and Gang Huang .Knockdown of RCAS1 expression by RNA interference recovers T cell growth and proliferation.Cancer Letters, Volume257, Issue 2, 18 November 2007, 182-190.
9. Alberto Carbonell,ángel-Emilio Martínez de Alba, Ricardo Flores ,et al.Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families.Virology, 29 October 2007,843-857.
10. Liu JD , Carmell A , Rivas V , et al . Argonaute2 Is the Catalytic Engine of Mammalian RNAi [ J ] . Science , 2004 , 305 ( 5689) : 1437 - 1441.
11. Lan J ,Zhou KH ,Li F , et al . Structural Basis for Double - Strand2 ed RNA Processing by Dicer[J ] . Science , 2006 ,311 (13) :195 -198.
12. Song JJ , Smith SK, Hannon GJ , et al . Crystal Structure of Arg2 onaute and Its Implications for RISC Slicer Activity[J ] . Science , 2004 ,305 (5689) :1434 - 1437.
13. Hammond SM ,Kobayashi R , Hannon GJ , et al . Argonaute2 , a Link Between Genetic and Biochemical Analyses of RNAi. Sci2 ence , 2001 ,293 (5532) :46 -50.
14. Sijen T , Simmer F , Fire A. et al . On the role of RNA amplifica2 tion in dsRNA - triggered gene silencing[J ] . Cell ,2001 ,107 (4) : 65 - 76.
15. Lipardi C , Wei Q , Paterson BM. RNAi as random degradative PCR : siRNA primers convert mRNA into dsRNAs that are de2 graded to generate new siRNAs . Cell ,2001 ,107 (3) :297 -307.
16. Parrish S,Fleenor J,Xu S,et al.Functional anatomy of a dsRNA trigger differential requirement for the two trigger strands in RNA interference.Mol cell,2000,6(5):1077-1087.
17. Elbashir SM,Martinez J,Patkaniow ska A,et al.Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate.EMBO J,2001,20(23):6877-6888.
18. Elbashir SM,Harborth J,Lendecke W,et al.Duplexes of 21 mucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature,2001, 411(6836): 494-498
19. Leung R K,Whittaker P A.RNA interference:from gene silencing to gene-specific therapeutics.Phamacol Ther,2005,107(2):222-239
1. Pascal Batard, Martin Jordan and Florian Wurm.Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection.Gene, Volume 270, Issues 1-2, 30 May 2001, Pages 61-68
2. Kadriye Ciftci and Robert J. Levy.Enhanced plasmid DNA transfection with lysosomotropic agents in cultured fibroblasts.International Journal of Pharmaceutics, Volume 218, Issues 1-2, 7 May 2001, Pages 81-92
3. Magdalena Guerra-Crespo, Jean-Louis Charli, Víctor Hugo Rosales-García .Polyethylenimine improves the transfection efficiency of primary cultures of post-mitotic rat fetal hypothalamic neurons.Journal of Neuroscience Methods, Volume 127, Issue 2, 15 August 2003, Pages 179-192
4. Tsunehito Higashi, Eiji Nagamori, Takefumi Sone .A novel transfection method for mammalian cells using calcium alginate microbeads.Journal of Bioscience and Bioengineering, Volume 97, Issue 3, 2004, Pages 191-195
5. Volker Oberle, Gary de Jong, Jan I.Efficient transfer of chromosome-based DNA constructs into mammalian cells.Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, Volume 1676, Issue 3, 20 February 2004, Pages 223-230
6. Jeong Ah Kim, Keunchang Cho, Young Shik Shin.A multi-channel electroporation microchip for gene transfection in mammalian cells.Biosensors and Bioelectronics, Volume 22, Issue 12, 15 June 2007, Pages 3273-3277
7. Sharon E. Reed, Elizabeth M. Staley.Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors.Journal of Virological Methods, Volume 138, Issues 1-2, December 2006, Pages 85-98
8. Robert M. Brazas and James E. Hagstrom .Delivery of Small Interfering RNA to Mammalian Cells in Culture by Using Cationic Lipid Polymer-Based Transfection Reagents.Methods in Enzymology, Volume 392, 2005, Pages 112-124
9. Alyson L. Peel .Transfection of mammalian cells.Methods, Volume 33, Issue 2, June 2004, Pages 93-94
10. Simone Kaiser, Michal Toborek. Liposomem ediated high efficiency transfection of human endothelial cellls [ J ]. J Vasc Res, 2001; 38: 1332143.
11. Mayu Sugiyama, MitusoMatsuura, Yoshito Takeuchi. Possiblemech anism of polycation liposome ( PCL ) mediated gene transfer .Biochimica Et Biophysica Acta, 2004; 1660: 24230.
12. Bailey A L, Cullis P R. Membrane fusion with cationic Liposomes: effects of target membrane lip id composition. Biochemistry, 1997; 36: 1628-1634.
13. Waldman A S, Waldman B C. Stable transfection ofmammalian cells by syringe mediated mechanical loading ofDNA. Analytical Biochemistry, 1998; 258: 216-222.
14. Sandra K, Peter P, Ulrich C et al. Ultrasound enhancement of Liposome mediated cell transfection caused by cavitation effects. Ul2 trasoundMed Biol, 2000; 26: 897-903
15. C. E. Hellweg, C. Baumstark-Khan and G. Horneck.Enhanced green fluorescent protein as reporter protein for biomonitoring of cytotoxic effects in mammalian cells Analytica Chimica Acta, Volume 427, Issue 2, 26 January 2001, 191-199
16. K.H.S. Arun, C.L. Kaul and P. Ramarao. Green fluorescent proteins in receptor research: An emerging tool for drug discovery Journal of Pharmacological and Toxicological Methods, Volume 51, Issue 1, January-February 2005, 1-23.
17. Yao-Chi Chung, Hung-Jen Liu and Yu-Chen Hu.Facile monitoring of avian reovirusσB expression and purification processes by tagged green fluorescent protein Enzyme and Microbial Technology, Volume 35, Issues 6-7, 1 December 2004, 494-500
18. Agnelo Furtado and Robert Henry. Measurement of green fluorescent protein concentration in single cells by image analysis.Analytical Biochemistry, Volume 310, Issue 1, 1 November 2002, 84-92
19. Daniel Hechler, Robert Nitsch .Green-fluorescent-protein-expressing mice as models for the study of axonal growth and regeneration in vitro.Brain Research Reviews, Volume 52, Issue 1, 30 August 2006, 160-169
1. Alan Hall. Rho GTPases and the Actin Cytoskeleton. Science 1998;279:509-514
2. Barbara Niederost, Thomas Oertle, Jens Fritsche, et al. Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Rac1.Neurosci 2002,22(23):10368-10376
3. Pier Paolo Di Fiore and Giorgio Scita. Eps8 in the midst of GTPases The International Journal of Biochemistry & Cell Biology, Volume 34, Issue 10, October 2002, Pages 1178-1183.
4. Nina Offenh?user, Daniela Castelletti, Lisa Mapelli,et al.Increased Ethanol Resistance and Consumption in Eps8 Knockout Mice Correlates with Altered Actin Dynamics.Cell, Volume 127, Issue 1, 6 October 2006, Pages 213-226
5. Metello Innocenti,Emanuela Frittoli,Isabella Ponzanelli,et al.Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abil,and Sos-1.The Journal of Cell Biology,2005,160;1,17-23
6. Masoud Shekarabi, Timothy E. K. The Netrin-1 Receptor DCC Promotes Filopodia Formation and Cell Spreading by Activating Cdc42 and Rac1.Mol Cell Neurosci 2002;19:1-17
7. Jaimie F. Borisoff, Carmen C.M. Chan,Gordon W. Hiebert, et al. Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates. Mol Cell Neurosci 2003;22:405-416
8. Patricia Kunda, Gavin Craig,Veronica Dominguez,et al. Abi,Sral, and Kette control the stability and location of SCAR/WAVE to regulate the formation of actin-baseded protrusions. Curr Biol,2003;13:1867-1875
9. Fan PD, Goff SP. Abl interactor 1 binds to sos and inhibits epidermal growth factor and v-Abl-induced activation of extracellular signal-regulated kinases. Mol Cell Biol2000;20(20):7591-7601
10. Courtney KD, Grove M, Vandongen H, Vandongen A, et al. Localization and phosporylation of Abl-interactor proteins, Abi1 and Abi2, in the developing nervous system. Mol Cell Neurosci 2000;16(3):244-257
11. Giorgio Scita, Pierluigi Tenca, Liliana B. Areces, et al. An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.JCB 2001;154(5): 1031-1044
12. Innocenti M, Frittoli E, Ponzanelli I,et al. Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abil, and Sos-1.JCB 2003;160(1)17-23
13. Steffen A, Rottner K,Ehinger J, et al. Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation. EMBO J 2004;23(4):749-759
14. Veronika Jenei,Tommy Andersson,Judit Jakus,et al.E3B1,a human homologue of the mous gene product Abi-1,sensitizes activation of Rap1 in response to epidermal growth factor.Experimental Cell Research 2005;310(2):463-473
15. M.Innocenti,P.Tenca,E.Frittoli,I.Ponzanelli,J.R.Falck,S.M.Brachmann,P.P.Di Fiore and G.Scita,Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abi1,and sos-1,J.Cell Biol. 2003,160(3):17-23
16. Touch and go: guidance cues signal to the growth cone cytoskeleton Current Opinion in Neurobiology, Volume 15, Issue 5, October 2005, Pages 521-526 Katherine Kalil and Erik W Dent
17. A. Disanza, M.F. Carlier, T.E. Stradal, et al.Eps8 controls actin-based motility by capping the barbed ends of actin filaments, Nat. Cell Biol. 6 (2004), pp. 1180–1188
18. N. Offenh?user, A. Borgonovo, A. Disanza, et al.The eps8 family of proteins links growth factor stimulation to actin reorganization generating functional redundancy in the Ras/Rac pathway, Mol. Biol. Cell 15 (2004), pp. 91–98
1 Bernstein E, Denli AM, Hannon GJ. The rest is silence. RNA,2001,7(11):1509-1521
2 Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans. Nature,1998,391(6669):806-8111
3 Wianny F. and Zernicka-Goetz M.,et al.Specific interference with gene function by double-stranded RNA in early mouse development.Nature Cell Biology 2000 2:2 :70-75
4 Brummelkamp TR,Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science,2002,296(5567):550-553
5 Bass BL. Double-stranded RNA as a template for gene silencing. Cell,2000, 101(3): 235-238
6 Gura T. A silence that speaks volumes. Nature, 2000, 404(6780): 804-808
7 Bobinnec Y., Fukuda M. and Nishida E.Identification and characterization of Caenorhabditis elegansγ-tubulin in dividing cells and differentiated tissues.Journal of Cell Science 2000 113:21 :3747-3759
8 Milhavet O., Gary D.S. and Mattson M.P.RNA Interference in Biology and Medicine . Pharmacological Reviews 2003 55:4 629-648
9 Arendt C.W., Tang G. and Zilberstein A.Vector Systems for the Delivery of Small Interfering RNAs: Managing the RISC.ChemBioChem 2003 4:11:1129-1136
10 Trejo-ávila L., Elizondo-González R., Trujillo-Murillo K.D.C.Antiviral therapy: Inhibition of hepatitis C virus expression by RNA interference directed against the NS5B region of the viral genome. Annals of Hepatology 2007 6:3 :174-180
11 Liu Y.P., Haasnoot J. and Berkhout B.Design of extended short hairpin RNAs for HIV-1 inhibition.Nucleic Acids Research 2007 35:17 :5683-5693
12 Carthew R.W .RNA interference: The fragile X syndrome connection.Curr. Biol. 2002 12:24 :852-R854
13 Dong-Dong L.Small interfering RNA (siRNA) inhibited human liver cancer cell line SMMC7721 proliferation and tumorigenesis.Hepato-Gastroenterology 2007 54:78 : 1731-1735
14 Wilda M, Fuchs U, Wossmann W,et al. Killing of leukemic cells with a BCR/ABL fusion gene by RNAi. Oncogene, 2002,21:5716-5725
15 Kunigal S., Lakka S.S., Gondi C.S., et al.RNAi-mediated downregulation of urokinase plasminogen activator receptor and matrix metalloprotease-9 in human breast cancer cells results in decreased tumor invasion, angiogenesis and growth.International Journal of Cancer 2007 121:10:2307-2316
16 Wu X., Fan J., Wang X.,et al.Downregulation of CCR1 inhibits human hepatocellular carcinoma cell invasion .Biochemical and Biophysical Research Communications 2007 355:4 :866-871
17 Marco Domeniconi, NiccolòZampieri, Tim Spencer,et al.MAG Induces Regulated Intramembrane Proteolysis of the p75 Neurotrophin Receptor to Inhibit Neurite Outgrowth.Neuron, Volume 46, Issue 6, 16 June 2005, 849-855
18 Lixin Fan, Saulius Girnius,Bruce Oakley,et al.Support of trigeminal sensory neurons by nonneuronal p75 neurotrophin receptors.Developmental Brain Research, Volume 150, Issue 1, 19 May 2004, Pages 23-39
19 Yuki Miyamoto, Junji Yamauchi, Atsushi Sanbe,et al. Dock6, a Dock-C subfamily guanine nucleotide exchanger, has the dual specificity for Rac1 and Cdc42 and regulates neurite outgrowth.Experimental Cell Research, Volume 313, Issue 4, 15 February 2007, Pages 791-804
20 Zubair Ahmed, Russell G. Dent, Ellen l. Disinhibition of neurotrophin-induced dorsal root ganglion cell neurite outgrowth on CNS myelin by siRNA-mediated knockdown of NgR, P75NTR and Rho-A. Molecular and Cellular Neuroscience,2005,28:509-523
21 Gao M.-Y., Xiao J.-D., Li Z.-Y.,et al.Inhibitory effect of RNA interference on the expression of interleukin-6 receptor in the acute phase of spinal cord injury. Journal of Clinical Rehabilitative Tissue Engineering Research 2007 11:32:6497-6501
1. Patricia Kunda,Gavin Craig,Veronica Dominguez,et al.Abi,Sral,and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions.Curr Biol 2006;13:1867-1875
2. Zubair Ahmed,Russell G.Dent,Ellen L.Disinhibition of neurotrophin-induced dorsal root ganglion cell neurite outgrowth on CNS myelin by siRNA-mediated knockdown of NgR.Mol Cell Neuroscience 2005;28(22):509-523
3. Masoud Shekarabi,Timothy E.K.The Netrin-1 Receptor DCC Promotes Filopodia formation and cell spreading by activating Cdc42 and Rac1.Mol Cell Neurosci 2004;19:1-17.
4. Paul A.Janmey.Creating a niche in the cytoskeleton:Actin reorganization by a protein kinase.PNAS 2002;98(26):14745-14747
5. Shi Y,Alin K,Goff S.P.Abl-interactor-1,a novel SH3 protein binding to the carboxy-terminal portion of the Abl protein,suppresses v-abl transforming activity.Genes Dev 1995;9(21):2583-2597.
6. Steffen A,Rottner K,Ehinger J,et al.Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation.EMBO J 2004;23(4):749-759
7. Giorgio Scita,Pierluigi Tenca,Liliana B.Areces,et al.An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.JCB 2005; 154 (5):1031-1044
8. Patricia Kunda,Gavin Craig,Veronica Dominguez,et al.Abi,Sral,and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions.Curr Biol 2005;13:1867-1875
9. Fan PD,Goff SP.Abl interactor 1 binds to sos and inhibits epidermal growth factor and v-Abl-induced activation of extracellular signal-regulated kinases.Mol Cell Biol 2005;20(20):7591-7601
10. Courtney KD,Grove M,Vandongen H,Vandongen A,et al.Localization and phosphorylation of Abl-interactor proteins,Abi1 and Abi-2,in the developing nevous system.Mol Cell Neurosci 2005;16(3):244-257
2. Liu BP, Fournier A, GrandPre T, et al. Myelin-Associated Glycoprotein as a Functional Ligand for the Nogo-66 Receptor. Science 2002;297(5584): 1190-1193.
3. Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature 2002;417(6892):941-944
4. Wang KC, Kim JA, Sivasankaran R, et al. p75 interacts with the Nogo receptor as a co-receptor for Nogo,MAG and OMgp. Nature 2002;420(6911):74-78.
5. Gentry JJ, Barker PA,Carter BD. The p75 neurotrophin receptor: multiple interactors and numerous functions. Prog Brain Res. 2004;146:25-39
6. Alan Hall. Rho GTPases and the Actin Cytoskeleton. Science 1998;279:509-514
7. Barbara Niederost, Thomas Oertle, Jens Fritsche, et al. Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Rac1.Neurosci 2002,22(23):10368-10376
8. Masoud Shekarabi, Timothy E. K. The Netrin-1 Receptor DCC Promotes Filopodia Formation and Cell Spreading by Activating Cdc42 and Rac1.Mol Cell Neurosci 2002;19:1-17
9. Jaimie F. Borisoff, Carmen C.M. Chan,Gordon W. Hiebert, et al. Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates. Mol Cell Neurosci 2003;22:405-416
10. Paul A. Janmey. Creating a niche in the cytoskeleton: Actin reorganization by a protein kinase. PNAS 2001;98(26):14745-14747
11. Dickson B.j. Rho in growth cone guidance. Curr Opin Neurobio 2001;11:103-110
12. Shi Y. Alin K. Goff S. P. Abl-interactor-1, a novel SH3 protein binding to the carboxy-terminal portion of the Abl protein, suppresses v-abl transforming activity.Genes Dev 1995;9(21):2583-2597
13. Giorgio Scita, Pierluigi Tenca, Liliana B. Areces, et al. An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.JCB2001;154(5):1031-1044
14. Innocenti M, Frittoli E, Ponzanelli I,et al. Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abil, and Sos-1.JCB 2003;160(1)17-23
15. Steffen A, Rottner K,Ehinger J, et al. Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation. EMBO J 2004;23(4):749-759
16. Patricia Kunda, Gavin Craig,Veronica Dominguez,et al. Abi,Sral, and Kette control the stability and location of SCAR/WAVE to regulate the formation of actin-baseded protrusions. Curr Biol2003;13:1867-1875
17. Fan PD, Goff SP. Abl interactor 1 binds to sos and inhibits epidermal growth factor and v-Abl-induced activation of extracellular signal-regulated kinases. Mol Cell Biol 2000;20(20):7591-7601
18. Courtney KD, Grove M, Vandongen H, Vandongen A, et al. Localization and phosporylation of Abl-interactor proteins, Abi1 and Abi2, in the developing nervous system. Mol Cell Neurosci 2000;16(3):244-257
19. Stradal T, Courtney KD, Rottner K, et al. The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia. Curr Biol 2001; 11(11):891-895
1. Gennadij Raivich and Milan Makwana .The making of successful axonal regeneration: Genes, molecules and signal transduction pathways Brain Research Reviews, Volume 53, Issue 2, February 2007, 287-311
2. Kuo-Cheng Huang, Zivart Yasruel, Claude Guérin ,et al.Interaction of the Coxsackie and adenovirus receptor (CAR) with the cytoskeleton: Binding to actinFEBS Letters, Volume 581, Issue 14, 12 June 2007, 2702-2708
3. Pramod K. Allani, Tak Sum, et al.A comparative study of the effect of oxidative stress on the cytoskeleton in human cortical neurons Toxicology and Applied Pharmacology, Volume 196, Issue 1, 1 April 2004, 29-36
4. Ching-Hui Huang, Ju-Chien Cheng, Jin-Chung,et al.Evaluation of the role of Disabled-2 in nerve growth factor-mediated neurite outgrowth and cellular signaling Cellular Signalling, Volume 19, Issue 6, June 2007, 1339-1347
5. Rachel de las Heras, Iris Depaz, Vincent Jaquet ,et al.Neuronal protein 22 colocalises with both the microtubule and microfilament cytoskeleton in neurite-like processes Brain Research, Volume 1128, 12 January 2007, 12-20
6. Bettina Büttner, Christoph Kannicht, Werner Reutter,et al.The neural cell adhesion molecule is associated with major components of the cytoskeleton Biochemical and Biophysical Research Communications, Volume 310, Issue 3, 24 October 2003, 967-971
7. E. Nyatia ,D.M. Lang Localisation and expression of a myelin associated neurite inhibitor, Nogo-A and its receptor Nogo-receptor by mammalian CNS cells Research in Veterinary Science, Volume 83, Issue 3, December 2007, 287-301
8. Oliver Weinmann, Lisa Schnell, Arko Ghosh,et al.Intrathecally infused antibodies against Nogo-A penetrate the CNS and downregulate the endogenous neurite growth inhibitor Nogo-A.Molecular and Cellular Neuroscience, Volume 32, Issues 1-2, May-June 2006, 161-173
9. Rüdiger Schweigreiter, Adrian R.,Walmsley,et al.Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA.Molecular and Cellular Neuroscience, Volume 27, Issue 2, 1 October 2004, 163-174
10. Ferdinando Rossi, Sara Gianola and Luigi Corvetti.Regulation of intrinsic neuronal properties for axon growth and regeneration.Progress in Neurobiology, Volume 81, Issue 1, January 2007, 1-28
11. H. Tanaka, T. Yamashita, K. Yachi,et al.Cytoplasmic p21Cip1/WAF1 enhances axonal regeneration and functional recovery after spinal cord injury in rats. Neurosc- ience, Volume 127, Issue 1, 2004, 155-164
12. Marco Domeniconi and Marie T. Filbin .Overcoming inhibitors in myelin to promote axonal regeneration.Journal of the Neurological Sciences, Volume 233, Issues 1-2, 15 June 2005, 43-47
13. Jennifer J. Gentry, Philip A,et al.The p75 neurotrophin receptor: multiple interactors and numerous functions.Progress in Brain Research, Volume 146, 2004, Pages 25-39
14. Altman J. Programmed cell death: the paths to suicide. Trends Neurosci, 1992, 15(8): 278-280.
15. Patrick Vourc'h and Christian Andres.Oligodendrocyte myelin glycoprotein (OMgp): evolution, structure and function.Brain Research Reviews, Volume 45, Issue 2, May 2004, 115-124
16. G.K.T. Chu, W. Yu,M.G. Fehlings.The p75 neurotrophin receptor is essential for neuronal cell survival and improvement of functional recovery after spinal cord injury.Neuroscience, Volume 148, Issue 3, 7 September 2007, Pages 668-682
17. Marco Domeniconi, NiccolòZampieri, Tim Spencer,et al.MAG Induces Regulated Intramembrane Proteolysis of the p75 Neurotrophin Receptor to Inhibit Neurite Outgrowth.Neuron, Volume 46, Issue 6, 16 June 2005, 849-855
18. Lixin Fan, Saulius Girnius,Bruce Oakley,et al.Support of trigeminal sensory neurons bynonneuronal p75 neurotrophin receptors.Developmental Brain Research, Volume 150, Issue 1, 19 May 2004, Pages 23-39
19. Yuki Miyamoto, Junji Yamauchi, Atsushi Sanbe,et al. Dock6, a Dock-C subfamily guanine nucleotide exchanger, has the dual specificity for Rac1 and Cdc42 and regulates neurite outgrowth.Experimental Cell Research, Volume 313, Issue 4, 15 February 2007, Pages 791-804
20. Charles E. Laurent and Thomas E. Smithgall.The c-Fes tyrosine kinase cooperates with the breakpoint cluster region protein (Bcr) to induce neurite extension in a Rac- and Cdc42-dependent manner.Experimental Cell Research, Volume 299, Issue 1, 10 September 2004, Pages 188-198
21. Denice L. Major,Susann M. Brady-Kalnay.Rho GTPases regulate PTPμ-mediated nasal neurite outgrowth and temporal repulsion of retinal ganglion cell neurons.Molecular and Cellular Neuroscience, Volume 34, Issue 3, March 2007, Pages 453-467
22. Anthony D. Couvillon and John H. Exton .Role of heterotrimeric G-proteins in lysophosphatidic acid-mediated neurite retraction by RhoA-dependent and -independent mechanisms in N1E-115 cells.Cellular Signalling, Volume 18, Issue 5, May 2006, Pages 715-728
23. Zubair Ahmed, Russell G. Dent, Ellen L ,et al.Disinhibition of neurotrophin-induced dorsal root ganglion cell neurite outgrowth on CNS myelin by siRNA-mediated knockdown of NgR, p75NTR and Rho-A.Molecular and Cellular Neuroscience, Volume 28, Issue 3, March 2005, Pages 509-523
24. Barbara Niederost, Thomas Oertle, Jens Fritsche, et al. Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Rac1.Neurosci 2002,22(23):10368-10376
25. Charles E. Laurent ,Thomas E. Smithgall The c-Fes tyrosine kinase cooperates with the breakpoint cluster region protein (Bcr) to induce neurite extension in a Rac- and Cdc42-dependent manner.Experimental Cell Research, Volume 299, Issue 1, 10 September 2004, Pages 188-198
26. Masoud Shekarabi, Timothy E. K. The Netrin-1 Receptor DCC Promotes Filopodia Formation and Cell Spreading by Activating Cdc42 and Rac1.Mol Cell Neurosci 2002;19:1-17
27. Jaimie F. Borisoff, Carmen C.M. Chan,Gordon W. Hiebert, et al. Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates. Mol Cell Neurosci 2003;22:405-416
28. Paul A. Janmey. Creating a niche in the cytoskeleton: Actin reorganization by a protein kinase. PNAS 2001;98(26):14745-14747
29. Dickson B.j. Rho in growth cone guidance. Curr Opin Neurobio 2001;11:103-110
30. Shi Y. Alin K. Goff S. P. Abl-interactor-1, a novel SH3 protein binding to the carboxy-terminal portion of the Abl protein, suppresses v-abl transforming activity.Genes Dev 1995;9(21):2583-2597
31. Giorgio Scita, Pierluigi Tenca, Liliana B. Areces, et al. An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and forthe proper localization of the Rac-based actin-polymerizing machine.JCB 2001; 154 (5):1031-1044
32. Innocenti M, Frittoli E, Ponzanelli I,et al. Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abil, and Sos-1.JCB 2003;160(1)17-23
33. Steffen A, Rottner K,Ehinger J, et al. Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation. EMBO J 2004;23(4):749-759
1. Falconer JC , Narayana PA , BhattacharieeM, et al . Characterization of an experiental spinal cord injury model suing waveformand morphometric analysis [ J ] . Spine , 1996 , 21 (1) : 104 - 112.
2. Khan T , Havey RM, Sayers ST , et al . Animal models of spinal cord contusion injuries [ J ] . Las Anim Sci , 1999 , 49 (2) : 161 - 172.
3. Hiruma S , Otsuka K, Satou T. et al . Simple and reproducible model of rat spinal cord injury induced by a controlled cortical impact device [J ] . Neurol Res , 1999 , 21 (3) :313 - 323.
4. Eugen J , Dolan , EnsorE , et al . The effect of spinal distraction on regional spinal cord blood flow in cats [J ] . J Neurosurg , 1980 , 53 (9) : 756 - 764.
5. Jarzem PF , Kostuik JP , Filiaggi M, et al . Spinal cord distraction : an vitro study of length , tension and tissue pressure [J ] . J Spine Disord , 1991 , 4 (2) : 177 - 182.
6. Maiman OJ , Myklebust JB , Ho Kc , et al . Experimental spinal cord injury produced by axial tension [J ] . J Spinal Disord , 1989 , 232 (1) : 6 - 13.
7. Allen AR,Surgery of Experimental Lesion of Spinal Cord Equivalent to Crush injury of fracture dislocation of spinal column.JAMA,1911,57:878-880.
8. Nystrom B , Berglund JE , Bergquist E. Methodological analysis of an experimentalspinal cord compression model in the rat [ J ] . J Acta Neurol Scand , 1988 , 78 (4) : 460 - 466.
9. Hashimoto T , FuKuta. New spinal cord injury model produced by spinal cord compression in the rat [ J ] . J pharmaol methods , 1990 , 23 (3) : 203 - 212.
10. Sato K, Konno S , Yabuki S , et al . Amodel for acute , chronic and delayed graded compression of the dog cauda equine [J ] . Spine , 1995 , 20 ( 22 ) : 2386 -2391.
11. Borgens RB. Functional recovery after spinal hemisection in guinea pigs the effects of applied electric field [ J ] . J Comp Neurol , 1990 , 96 (6) : 634 - 640.
12. Colak A , Nurlu G, Acikgoz B , et al . a new experimental model for further investigation of the secondary changes following spinal cord injury [ J ] . J Neurosurg Sci , 1994 , 38 (2) : 111 - 116.
13. Lo YC , Mcbrie WH , Wither HR. The effect of single doses of radiation on mouse spinal cord [ J ] . Int J Radiat Oncol Biol Phys , 1992 , 22 (1) : 57 - 63.
14. Tarlov IM, Klinger H. Spinal cord compression studies I. Experimental techniques to produce acute and gradual compression [J ] . J A M A. Arch , Neurol &Psychiat , 1953 , 70 (7) : 813 - 819.
15. Tator CH, Fehlings LG. Review of the secordary injury theory of acute spinal cord trauma with emphasis on vascular mechanism [J ]. J Newrosurg , 1991 , 75 (1) : 15 - 26.
1. Oliver Weinmann, Lisa Schnell, Arko Ghosh,et al.Intrathecally infused antibodies against Nogo-A penetrate the CNS and downregulate the endogenous neurite growth inhibitor Nogo-A.Molecular and Cellular Neuroscience, Volume 32, Issues 1-2, May-June 2006, 161-173
2. Rüdiger Schweigreiter, Adrian R.,Walmsley,et al.Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA.Molecular and Cellular Neuroscience, Volume 27, Issue 2, 1 October 2004, 163-174
3. H. Tanaka, T. Yamashita, K. Yachi,et al.Cytoplasmic p21Cip1/WAF1 enhances axonal regeneration and functional recovery after spinal cord injury in rats. Neuroscience, Volume 127, Issue 1, 2004, 155-164
4. Marco Domeniconi and Marie T. Filbin .Overcoming inhibitors in myelin to promoteaxonal regeneration.Journal of the Neurological Sciences, Volume 233, Issues 1-2, 15 June 2005, 43-47
5. Altman J. Programmed cell death: the paths to suicide. Trends Neurosci, 1992, 15(8): 278-280.
6. Patrick Vourc'h and Christian Andres.Oligodendrocyte myelin glycoprotein (OMgp): evolution, structure and function.Brain Research Reviews, Volume 45, Issue 2, May 2004, 115-124
7. Philippe P. Monnier, Ana Sierra,et al.The Rho/ROCK pathway mediates neurite growth-inhibitory activity associated with the chondroitin sulfate proteoglycans of the CNS glial scar.Molecular and Cellular Neuroscience, Volume 22, Issue 3, March 2003, 319-330
8. Diane M. Snow, Jeffrey D. Smith ,et al.Neurite elongation on chondroitin sulfate proteoglycans is characterized by axonal fasciculation.Experimental Neurology, Volume 182, Issue 2, August 2003, Pages 310-321
9. Li S,Strittmatter SM.Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury.Neurosci,2003,23(10):4219-4227.
10. Li SX,Liu BP,Budel S.Blockade of Nogo-66,myelin-associated glycoprotein,and oligodendrocyte myelin glyeoprotein by soluble Nogo-66 receptor promotes axonal sprouting and after spinal injury.Neuroscience,2004,24(46):10511-10520.
11. Ruitenberg M J,Levison DB,Lee SV,et al.Nt-3expression from engineered olfactory ensheathing glia promotes spinal sparing and regeneration.Brain, 2005,128(4): 839-853.
12. Xu G,Nie DY,Chen JT,et al.Recombinant DNA vaceine encoding multiple domains related to inhibition of neurite outgroeth:a potentialstrategy for axonal regeneration.Journal of neurochemistry.2004,91(4):1018-1023
13. Jaimie F.Borisoff,Carmen C.M.Chan,Gordon W.Hiebert,et al.Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates.Mol Cell Neurosci 2003;22:405-416
14. Patricia Kunda,Gavin Craig,Veronica Dominguez,et al.Abi,Sral,and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions.Curr Biol 2003;13:1867-1875
15. Courtney KD,Grove M,Vandongen H,Vandongen A,et al.Localization and phosphorylation of Abl-interactor proteins,Abi1 and Abi-2,in the developing nevous system.Mol Cell Neurosci 2002;16(3):244-257
16. Stradal T,Courtney KD,Rottner K,et al.The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia.Curr Biol 2002;11(11):891-895
17. Paul A.Janmey.Creating a niche in the cytoskeleton:Actin reorganization by a protein kinase.PNAS 2002;98(26):14745-14747
18. Innocenti M,Frittoli E,Ponzanelli I,et al.Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abi1,and Sos-1.JCB 2003;160(1):17-23
19. Veronika Jenei,Tommy Andersson,Judit Jakus,et al.E3B1,a human homologue of the mous gene product Abi-1,sensitizes activation of Rap1 in response to epidermal growth factor.Experimental Cell Research 2005;310(2):463-473
20. M.Innocenti,P.Tenca,E.Frittoli,I.Ponzanelli,J.R.Falck,S.M.Brachmann,P.P.Di Fiore and G.Scita,Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abi1,and sos-1,J.Cell Biol.160(2003),PP.17-23
21. Patricia Kunda,Gavin Craig,Veronica Dominguez,et al.Abi,Sral,and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions.Curr Biol 2003;13:1867-1875
22. Steffen A,Rottner K,Ehinger J,et al.Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation.EMBO J 2004;23(4):749-759
1. Kuhn TB. Growing and working with spinal motor neurons. Methods Cell Biol, 2003, 71(1): 67-97.
2. Van Damme P, Van Den BL, Van Houtte E, et al. Na+ entry through AMPA receptors results in voltage-gated K+ channel blockade in cultured rat spinal cord motoneurons. J Neurophysiol. 200, 88(2): 965-972.
3. Guigoni C, Coulon P. Rabies virus is not cytolytic for rat spinal motoneurons in vitro. J Neurovirol, 2002, 8(4): 306-317.
4. Mouveroux JM, Lakke EA, Marani E. Lumbar spinal cord explants from neonatal rat display age-related decrease of outgrowth in culture. Neurosci Lett, 2001, 311(2): 69-72.
5. Erkman L, Touzeau G, Bertrand D, et al. Characterization of dissociated monolayer cultures of human spinal cord. Brain Research Bulletin, 1989, 22: 57-65.
6. Michikawa M, Lim KT, McLarnon JG, et al. Oxygen radical induced neurotoxicity in spinal cord neuron cultures. Neurosci Res, 1994, 37: 62-70.
7. Andersona KN, Potterb AC, Piccenna LG, et al. Isolation and culture of motor neurons from the newborn mouse spinal cord. Brain Research Protocols, 2004, 12: 132– 136.
8. Vejsada R, Tseng JL, Lindsay RM, et al. Synergistic but transient rescue effects of BDNF and GDNF on axotomized neonatal motoneurons. Neuroscience, 1998, 84(1): 129-139.
9. Chen B, Hammonds-Odie L, Perron J, et al. SHP-2 Mediates Target-Regulated Axonal Termination and NGF-Dependent Neurite Growth in Sympathetic Neurons. Dev Biol, 2002, 252(2): 170-187.
10. Lindholm D, Mercer EA, Yu LY, et al. Neuronal apoptosis inhibitory protein: structural requirements for hippocalcin binding and effects on survival of NGF-dependent sympathetic neurons. BBA-Proteins Proteomics, 2002, 1600(1-2): 138-147.
11. Brian A, Pierchala T, ilbrandt J, et al. NGF Utilizes c-Ret Via a Novel GFL–Independent, Inter-RTK Signaling Mechanism to Maintain the Trophic Status of Mature Sympathetic Neurons. Neuron, 2002, 33(2): 261-273.
12. Bataille S, Portalier P, Coulon P, et al. Influence of acetylcholinesterase on embryonic spinal rat motoneurons growth in culture: a quantitative mophometric study. Eur J Neurosci, 1998, 10: 560-572.
13. Camu W and Henderson, CE. Purification of embryonic rat motoneurons by panning on a monoclonal antibody to the low affinity NGF receptor. J Neurosci Methods, 1992, 44(1): 59-70.
14. Altman J. Programmed cell death: the paths to suicide. Trends Neurosci, 1992, 15(8): 278-280.
15. Oppenheim RW. Cell death during development of the nervous system. Annu Rev Neurosci, 1991, 14: 453-501.
16. Sendtner M, Pei G, Beck M, et al. Developmental motoneuron cell death and neurotrophic factors. Cell Tissue Res, 2000, 301: 71–84.
17. Haastert K, Grosskreutz J, Jaeckel M, et al. Rat embryonic motoneurons in long-term co-culture with Schwann cells—a system to investigate motoneuron diseases on a cellular level in vitro. J Neurosci Methods, 2005, 142: 275–284.
18. Martinou IS, Desagher R, Eskes B, et al. The release of cytochrome c from mitochondria during apoptosis of NGF-deprived sympathetic neurons is a reversible event. J Cell Biol, 1999, 144(5): 883–889.
19. Brodski C, Schaubmar A, Dechant G. Opposing functions of GDNF and NGF in the development of cholinergic and noradrenergic sympathetic neurons. Mol Cel Neurosci, 2002, 19(4): 528-538.
20. Henderson CE, Phillips HS, Pollock RA, et al. GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle[J]. Science, 1994,266(5187): 1062-1064.
21. Carriedo SG, Sensi SL, Yin HZ, et al. AMPA exposure induce mitochondrial Ca2+ overload and ROS generation in spinal motor neurons in vitro. J Neurosci, 2000, 20(1): 240-250.
22. Roy J, Minotti S, Dong L, et al. Glutamate potentiates the toxicity of mutant Cu/Zn-superoxide dismutase in motor neurons by postsynaptic calcium-dependentmechanism. J Neurosci, 1998, 18(23): 9673-9684.
1. H. Tanaka, T. Yamashita, K. Yachi,et al.Cytoplasmic p21Cip1/WAF1 enhances axonal regeneration and functional recovery after spinal cord injury in rats. Neuroscience, Volume 127, Issue 1, 2004, 155-164
2. Marco Domeniconi and Marie T. Filbin .Overcoming inhibitors in myelin to promote axonal regeneration.Journal of the Neurological Sciences, Volume 233, Issues 1-2, 15 June 2005, 43-47
3. Altman J. Programmed cell death: the paths to suicide. Trends Neurosci, 1992, 15(8): 278-280.
4. Patrick Vourc'h and Christian Andres.Oligodendrocyte myelin glycoprotein (OMgp): evolution, structure and function.Brain Research Reviews, Volume 45, Issue 2, May 2004, 115-124
5. Philippe P. Monnier, Ana Sierra,et al.The Rho/ROCK pathway mediates neurite growth-inhibitory activity associated with the chondroitin sulfate proteoglycans of the CNS glial scar.Molecular and Cellular Neuroscience, Volume 22, Issue 3, March 2003, 319-330
6. Diane M. Snow, Jeffrey D. Smith ,et al.Neurite elongation on chondroitin sulfate proteoglycans is characterized by axonal fasciculation.Experimental Neurology, Volume 182, Issue 2, August 2003, Pages 310-321
7. Monnier PP , Sierra A , Schwab JM, et al. The Rho/ ROCK pathway mediates neurite growth2inhibitory activity associated with the chondroitin sulfate proteoglycans of the CNS glial scar. Mol Cell Neurosci , 2003 , 22÷319～330.
8. Emilie Pacary, Emmanuelle Tixier, Florence Coulet ,et al.Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth.Molecular and Cellular Neuroscience, Volume 35, Issue 3, July 2007, Pages 409-423
9. Philippe P. Monnier, Ana Sierra,The Rho/ROCK pathway mediates neurite growth-inhibitory activity associated with the chondroitin sulfate proteoglycans of the CNS glial scar.Molecular and Cellular Neuroscience, Volume 22, Issue 3, March 2003, Pages 319-330
10. Rüdiger Schweigreiter, Adrian R ,et al.Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA.Molecular and Cellular Neuroscience, Volume 27, Issue 2, 1 October 2004, Pages 163-174
11. Changman Zhou, Yun Li .HBO suppresses Nogo-A, Ng-R, or RhoA expression in the cerebral cortex after global ischemia.Biochemical and Biophysical Research Communications, Volume 309, Issue 2, 19 September 2003, Pages 368-376
12. Binhai Zheng, Carole Ho, Shuxin Li .Lack of Enhanced Spinal Regeneration in Nogo-Deficient MiceNeuron, Volume 38, Issue 2, 24 April 2003, Pages 213-224
13. Aaron W. McGee and Stephen M. Strittmatter .The Nogo-66 receptor: focusing myelin inhibition of axon regeneration.Trends in Neurosciences, Volume 26, Issue 4, April 2003, Pages 193-198
14. Wang KC, Kim JA, Sivasankaran R, et al. p75 interacts with the Nogo receptor as a co-receptor for Nogo,MAG and OMgp. Nature 2002;420(6911):74-78.
15. Gentry JJ, Barker PA,Carter BD. The p75 neurotrophin receptor: multiple interactors and numerous functions. Prog Brain Res. 2004;146:25-39
16. Alan Hall. Rho GTPases and the Actin Cytoskeleton. Science 1998;279:509-514
17. Zubair Ahmed,Russell G,Dent,et al.Disinhibition of neurotrophin-induced dorsal root ganglion cell neurite outgrowth on CNS nyelin by siRNA-mediated knockdown of NgR,p75NTR and Rho-A.Mol.Cell.Neurosci.2005,28,509-523.
1. Ng CE, Tang BL.Nogos and the Nogo-66 receptor: factors inhibiting CNS neuron regeneration. J Neurosci Res 2002;67(5):559-565
2. Liu BP, Fournier A, GrandPre T, et al. Myelin-Associated Glycoprotein as a Functional Ligand for the Nogo-66 Receptor. Science 2002;297(5584): 1190-1193.
3. Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature 2002;417(6892):941-944
4. Wang KC, Kim JA, Sivasankaran R, et al. p75 interacts with the Nogo receptor as a co-receptor for Nogo,MAG and OMgp. Nature 2002;420(6911):74-78.
5. Gentry JJ, Barker PA,Carter BD. The p75 neurotrophin receptor: multiple interactors and numerous functions. Prog Brain Res. 2004;146:25-39
6. Alan Hall. Rho GTPases and the Actin Cytoskeleton. Science 1998;279:509-514
7. Barbara Niederost, Thomas Oertle, Jens Fritsche, et al. Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Rac1.Neurosci 2002,22(23):10368-10376
8. Masoud Shekarabi, Timothy E. K. The Netrin-1 Receptor DCC Promotes Filopodia Formation and Cell Spreading by Activating Cdc42 and Rac1.Mol Cell Neurosci 2002;19:1-17
9. Jaimie F. Borisoff, Carmen C.M. Chan,Gordon W. Hiebert, et al. Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates. Mol Cell Neurosci 2003;22:405-416
10. Paul A. Janmey. Creating a niche in the cytoskeleton: Actin reorganization by a protein kinase. PNAS 2001;98(26):14745-14747
11. Dickson B.j. Rho in growth cone guidance. Curr Opin Neurobio 2001;11:103-110
12. Shi Y. Alin K. Goff S. P. Abl-interactor-1, a novel SH3 protein binding to the carboxy-terminal portion of the Abl protein, suppresses v-abl transforming activity.Genes Dev 1995;9(21):2583-2597
13. Giorgio Scita, Pierluigi Tenca, Liliana B. Areces, et al. An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.JCB 2001;154(5):1031-1044
14. Innocenti M, Frittoli E, Ponzanelli I,et al. Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abil, and Sos-1.JCB 2003;160(1)17-23
15. Steffen A, Rottner K,Ehinger J, et al. Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation. EMBO J 2004;23(4):749-759
16. Kazuhiro Ishiguro, Zhifang Cao, Marco Lopez Ilasca ,et al.Wave2 activates serum response element via its VCA region and functions downstream of Rac.Experimental Cell Research, Volume 301, Issue 2, 10 December 2004, Pages 331-337.
17. Patricia Kunda, Gavin Craig,Veronica Dominguez,et al. Abi,Sral, and Kette control the stability and location of SCAR/WAVE to regulate the formation of actin-baseded protrusions. Curr Biol2003;13:1867-1875
18. Fan PD, Goff SP. Abl interactor 1 binds to sos and inhibits epidermal growth factor and v-Abl-induced activation of extracellular signal-regulated kinases. Mol Cell Biol 2000;20(20):7591-7601
19. Courtney KD, Grove M, Vandongen H, Vandongen A, et al. Localization and phosporylation of Abl-interactor proteins, Abi1 and Abi2, in the developing nervous system. Mol Cell Neurosci 2000;16(3):244-257
20. Stradal T, Courtney KD, Rottner K, et al. The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia. Curr Biol 2001;11(11):891-895
1. Fire A,Xu S,Montgomery M K,et al.Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans.Nature,1998,391(6669):806-811.
2. Bernstin E,Caudy AA,Hammond SM,et al.Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature,2001,409 (6818):363-366.
3. Hannon GJ.RNA interference.Nature,2002,418(6894):244-251
4. Zeng Y,Cullen Br.RNA interference in human cells is restricted to the cytoplasm.RNA,2002,8:855-867
5. Zamore PD.RNA interference:listening to the sound of silence.Nat Struct Biol,2001,8(9):746-750
6. Zheng ZM , Tang S , Tao Mf. Development of Resistance to RNAi in Mammalian Cells[J ] . Science , 2005 ,1058 : 105 - 118.
7. Makoto Inaki, Takahiko Utsugi, Fumitoshi Onoda ,et al.A novel class of temperature-sensitive mutants generated by RNAi-mediated knockdown. Biochemical and Biophysical Research Communications, Volume 365, Issue 3, 18 January 2008, 472-477
8. Yuan Han, Wenxin Qin and Gang Huang .Knockdown of RCAS1 expression by RNA interference recovers T cell growth and proliferation.Cancer Letters, Volume257, Issue 2, 18 November 2007, 182-190.
9. Alberto Carbonell,ángel-Emilio Martínez de Alba, Ricardo Flores ,et al.Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families.Virology, 29 October 2007,843-857.
10. Liu JD , Carmell A , Rivas V , et al . Argonaute2 Is the Catalytic Engine of Mammalian RNAi [ J ] . Science , 2004 , 305 ( 5689) : 1437 - 1441.
11. Lan J ,Zhou KH ,Li F , et al . Structural Basis for Double - Strand2 ed RNA Processing by Dicer[J ] . Science , 2006 ,311 (13) :195 -198.
12. Song JJ , Smith SK, Hannon GJ , et al . Crystal Structure of Arg2 onaute and Its Implications for RISC Slicer Activity[J ] . Science , 2004 ,305 (5689) :1434 - 1437.
13. Hammond SM ,Kobayashi R , Hannon GJ , et al . Argonaute2 , a Link Between Genetic and Biochemical Analyses of RNAi. Sci2 ence , 2001 ,293 (5532) :46 -50.
14. Sijen T , Simmer F , Fire A. et al . On the role of RNA amplifica2 tion in dsRNA - triggered gene silencing[J ] . Cell ,2001 ,107 (4) : 65 - 76.
15. Lipardi C , Wei Q , Paterson BM. RNAi as random degradative PCR : siRNA primers convert mRNA into dsRNAs that are de2 graded to generate new siRNAs . Cell ,2001 ,107 (3) :297 -307.
16. Parrish S,Fleenor J,Xu S,et al.Functional anatomy of a dsRNA trigger differential requirement for the two trigger strands in RNA interference.Mol cell,2000,6(5):1077-1087.
17. Elbashir SM,Martinez J,Patkaniow ska A,et al.Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate.EMBO J,2001,20(23):6877-6888.
18. Elbashir SM,Harborth J,Lendecke W,et al.Duplexes of 21 mucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature,2001, 411(6836): 494-498
19. Leung R K,Whittaker P A.RNA interference:from gene silencing to gene-specific therapeutics.Phamacol Ther,2005,107(2):222-239
1. Pascal Batard, Martin Jordan and Florian Wurm.Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection.Gene, Volume 270, Issues 1-2, 30 May 2001, Pages 61-68
2. Kadriye Ciftci and Robert J. Levy.Enhanced plasmid DNA transfection with lysosomotropic agents in cultured fibroblasts.International Journal of Pharmaceutics, Volume 218, Issues 1-2, 7 May 2001, Pages 81-92
3. Magdalena Guerra-Crespo, Jean-Louis Charli, Víctor Hugo Rosales-García .Polyethylenimine improves the transfection efficiency of primary cultures of post-mitotic rat fetal hypothalamic neurons.Journal of Neuroscience Methods, Volume 127, Issue 2, 15 August 2003, Pages 179-192
4. Tsunehito Higashi, Eiji Nagamori, Takefumi Sone .A novel transfection method for mammalian cells using calcium alginate microbeads.Journal of Bioscience and Bioengineering, Volume 97, Issue 3, 2004, Pages 191-195
5. Volker Oberle, Gary de Jong, Jan I.Efficient transfer of chromosome-based DNA constructs into mammalian cells.Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, Volume 1676, Issue 3, 20 February 2004, Pages 223-230
6. Jeong Ah Kim, Keunchang Cho, Young Shik Shin.A multi-channel electroporation microchip for gene transfection in mammalian cells.Biosensors and Bioelectronics, Volume 22, Issue 12, 15 June 2007, Pages 3273-3277
7. Sharon E. Reed, Elizabeth M. Staley.Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors.Journal of Virological Methods, Volume 138, Issues 1-2, December 2006, Pages 85-98
8. Robert M. Brazas and James E. Hagstrom .Delivery of Small Interfering RNA to Mammalian Cells in Culture by Using Cationic Lipid Polymer-Based Transfection Reagents.Methods in Enzymology, Volume 392, 2005, Pages 112-124
9. Alyson L. Peel .Transfection of mammalian cells.Methods, Volume 33, Issue 2, June 2004, Pages 93-94
10. Simone Kaiser, Michal Toborek. Liposomem ediated high efficiency transfection of human endothelial cellls [ J ]. J Vasc Res, 2001; 38: 1332143.
11. Mayu Sugiyama, MitusoMatsuura, Yoshito Takeuchi. Possiblemech anism of polycation liposome ( PCL ) mediated gene transfer .Biochimica Et Biophysica Acta, 2004; 1660: 24230.
12. Bailey A L, Cullis P R. Membrane fusion with cationic Liposomes: effects of target membrane lip id composition. Biochemistry, 1997; 36: 1628-1634.
13. Waldman A S, Waldman B C. Stable transfection ofmammalian cells by syringe mediated mechanical loading ofDNA. Analytical Biochemistry, 1998; 258: 216-222.
14. Sandra K, Peter P, Ulrich C et al. Ultrasound enhancement of Liposome mediated cell transfection caused by cavitation effects. Ul2 trasoundMed Biol, 2000; 26: 897-903
15. C. E. Hellweg, C. Baumstark-Khan and G. Horneck.Enhanced green fluorescent protein as reporter protein for biomonitoring of cytotoxic effects in mammalian cells Analytica Chimica Acta, Volume 427, Issue 2, 26 January 2001, 191-199
16. K.H.S. Arun, C.L. Kaul and P. Ramarao. Green fluorescent proteins in receptor research: An emerging tool for drug discovery Journal of Pharmacological and Toxicological Methods, Volume 51, Issue 1, January-February 2005, 1-23.
17. Yao-Chi Chung, Hung-Jen Liu and Yu-Chen Hu.Facile monitoring of avian reovirusσB expression and purification processes by tagged green fluorescent protein Enzyme and Microbial Technology, Volume 35, Issues 6-7, 1 December 2004, 494-500
18. Agnelo Furtado and Robert Henry. Measurement of green fluorescent protein concentration in single cells by image analysis.Analytical Biochemistry, Volume 310, Issue 1, 1 November 2002, 84-92
19. Daniel Hechler, Robert Nitsch .Green-fluorescent-protein-expressing mice as models for the study of axonal growth and regeneration in vitro.Brain Research Reviews, Volume 52, Issue 1, 30 August 2006, 160-169
1. Alan Hall. Rho GTPases and the Actin Cytoskeleton. Science 1998;279:509-514
2. Barbara Niederost, Thomas Oertle, Jens Fritsche, et al. Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Rac1.Neurosci 2002,22(23):10368-10376
3. Pier Paolo Di Fiore and Giorgio Scita. Eps8 in the midst of GTPases The International Journal of Biochemistry & Cell Biology, Volume 34, Issue 10, October 2002, Pages 1178-1183.
4. Nina Offenh?user, Daniela Castelletti, Lisa Mapelli,et al.Increased Ethanol Resistance and Consumption in Eps8 Knockout Mice Correlates with Altered Actin Dynamics.Cell, Volume 127, Issue 1, 6 October 2006, Pages 213-226
5. Metello Innocenti,Emanuela Frittoli,Isabella Ponzanelli,et al.Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abil,and Sos-1.The Journal of Cell Biology,2005,160;1,17-23
6. Masoud Shekarabi, Timothy E. K. The Netrin-1 Receptor DCC Promotes Filopodia Formation and Cell Spreading by Activating Cdc42 and Rac1.Mol Cell Neurosci 2002;19:1-17
7. Jaimie F. Borisoff, Carmen C.M. Chan,Gordon W. Hiebert, et al. Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates. Mol Cell Neurosci 2003;22:405-416
8. Patricia Kunda, Gavin Craig,Veronica Dominguez,et al. Abi,Sral, and Kette control the stability and location of SCAR/WAVE to regulate the formation of actin-baseded protrusions. Curr Biol,2003;13:1867-1875
9. Fan PD, Goff SP. Abl interactor 1 binds to sos and inhibits epidermal growth factor and v-Abl-induced activation of extracellular signal-regulated kinases. Mol Cell Biol2000;20(20):7591-7601
10. Courtney KD, Grove M, Vandongen H, Vandongen A, et al. Localization and phosporylation of Abl-interactor proteins, Abi1 and Abi2, in the developing nervous system. Mol Cell Neurosci 2000;16(3):244-257
11. Giorgio Scita, Pierluigi Tenca, Liliana B. Areces, et al. An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.JCB 2001;154(5): 1031-1044
12. Innocenti M, Frittoli E, Ponzanelli I,et al. Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abil, and Sos-1.JCB 2003;160(1)17-23
13. Steffen A, Rottner K,Ehinger J, et al. Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation. EMBO J 2004;23(4):749-759
14. Veronika Jenei,Tommy Andersson,Judit Jakus,et al.E3B1,a human homologue of the mous gene product Abi-1,sensitizes activation of Rap1 in response to epidermal growth factor.Experimental Cell Research 2005;310(2):463-473
15. M.Innocenti,P.Tenca,E.Frittoli,I.Ponzanelli,J.R.Falck,S.M.Brachmann,P.P.Di Fiore and G.Scita,Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8,Abi1,and sos-1,J.Cell Biol. 2003,160(3):17-23
16. Touch and go: guidance cues signal to the growth cone cytoskeleton Current Opinion in Neurobiology, Volume 15, Issue 5, October 2005, Pages 521-526 Katherine Kalil and Erik W Dent
17. A. Disanza, M.F. Carlier, T.E. Stradal, et al.Eps8 controls actin-based motility by capping the barbed ends of actin filaments, Nat. Cell Biol. 6 (2004), pp. 1180–1188
18. N. Offenh?user, A. Borgonovo, A. Disanza, et al.The eps8 family of proteins links growth factor stimulation to actin reorganization generating functional redundancy in the Ras/Rac pathway, Mol. Biol. Cell 15 (2004), pp. 91–98
1 Bernstein E, Denli AM, Hannon GJ. The rest is silence. RNA,2001,7(11):1509-1521
2 Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans. Nature,1998,391(6669):806-8111
3 Wianny F. and Zernicka-Goetz M.,et al.Specific interference with gene function by double-stranded RNA in early mouse development.Nature Cell Biology 2000 2:2 :70-75
4 Brummelkamp TR,Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science,2002,296(5567):550-553
5 Bass BL. Double-stranded RNA as a template for gene silencing. Cell,2000, 101(3): 235-238
6 Gura T. A silence that speaks volumes. Nature, 2000, 404(6780): 804-808
7 Bobinnec Y., Fukuda M. and Nishida E.Identification and characterization of Caenorhabditis elegansγ-tubulin in dividing cells and differentiated tissues.Journal of Cell Science 2000 113:21 :3747-3759
8 Milhavet O., Gary D.S. and Mattson M.P.RNA Interference in Biology and Medicine . Pharmacological Reviews 2003 55:4 629-648
9 Arendt C.W., Tang G. and Zilberstein A.Vector Systems for the Delivery of Small Interfering RNAs: Managing the RISC.ChemBioChem 2003 4:11:1129-1136
10 Trejo-ávila L., Elizondo-González R., Trujillo-Murillo K.D.C.Antiviral therapy: Inhibition of hepatitis C virus expression by RNA interference directed against the NS5B region of the viral genome. Annals of Hepatology 2007 6:3 :174-180
11 Liu Y.P., Haasnoot J. and Berkhout B.Design of extended short hairpin RNAs for HIV-1 inhibition.Nucleic Acids Research 2007 35:17 :5683-5693
12 Carthew R.W .RNA interference: The fragile X syndrome connection.Curr. Biol. 2002 12:24 :852-R854
13 Dong-Dong L.Small interfering RNA (siRNA) inhibited human liver cancer cell line SMMC7721 proliferation and tumorigenesis.Hepato-Gastroenterology 2007 54:78 : 1731-1735
14 Wilda M, Fuchs U, Wossmann W,et al. Killing of leukemic cells with a BCR/ABL fusion gene by RNAi. Oncogene, 2002,21:5716-5725
15 Kunigal S., Lakka S.S., Gondi C.S., et al.RNAi-mediated downregulation of urokinase plasminogen activator receptor and matrix metalloprotease-9 in human breast cancer cells results in decreased tumor invasion, angiogenesis and growth.International Journal of Cancer 2007 121:10:2307-2316
16 Wu X., Fan J., Wang X.,et al.Downregulation of CCR1 inhibits human hepatocellular carcinoma cell invasion .Biochemical and Biophysical Research Communications 2007 355:4 :866-871
17 Marco Domeniconi, NiccolòZampieri, Tim Spencer,et al.MAG Induces Regulated Intramembrane Proteolysis of the p75 Neurotrophin Receptor to Inhibit Neurite Outgrowth.Neuron, Volume 46, Issue 6, 16 June 2005, 849-855
18 Lixin Fan, Saulius Girnius,Bruce Oakley,et al.Support of trigeminal sensory neurons by nonneuronal p75 neurotrophin receptors.Developmental Brain Research, Volume 150, Issue 1, 19 May 2004, Pages 23-39
19 Yuki Miyamoto, Junji Yamauchi, Atsushi Sanbe,et al. Dock6, a Dock-C subfamily guanine nucleotide exchanger, has the dual specificity for Rac1 and Cdc42 and regulates neurite outgrowth.Experimental Cell Research, Volume 313, Issue 4, 15 February 2007, Pages 791-804
20 Zubair Ahmed, Russell G. Dent, Ellen l. Disinhibition of neurotrophin-induced dorsal root ganglion cell neurite outgrowth on CNS myelin by siRNA-mediated knockdown of NgR, P75NTR and Rho-A. Molecular and Cellular Neuroscience,2005,28:509-523
21 Gao M.-Y., Xiao J.-D., Li Z.-Y.,et al.Inhibitory effect of RNA interference on the expression of interleukin-6 receptor in the acute phase of spinal cord injury. Journal of Clinical Rehabilitative Tissue Engineering Research 2007 11:32:6497-6501
1. Patricia Kunda,Gavin Craig,Veronica Dominguez,et al.Abi,Sral,and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions.Curr Biol 2006;13:1867-1875
2. Zubair Ahmed,Russell G.Dent,Ellen L.Disinhibition of neurotrophin-induced dorsal root ganglion cell neurite outgrowth on CNS myelin by siRNA-mediated knockdown of NgR.Mol Cell Neuroscience 2005;28(22):509-523
3. Masoud Shekarabi,Timothy E.K.The Netrin-1 Receptor DCC Promotes Filopodia formation and cell spreading by activating Cdc42 and Rac1.Mol Cell Neurosci 2004;19:1-17.
4. Paul A.Janmey.Creating a niche in the cytoskeleton:Actin reorganization by a protein kinase.PNAS 2002;98(26):14745-14747
5. Shi Y,Alin K,Goff S.P.Abl-interactor-1,a novel SH3 protein binding to the carboxy-terminal portion of the Abl protein,suppresses v-abl transforming activity.Genes Dev 1995;9(21):2583-2597.
6. Steffen A,Rottner K,Ehinger J,et al.Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation.EMBO J 2004;23(4):749-759
7. Giorgio Scita,Pierluigi Tenca,Liliana B.Areces,et al.An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.JCB 2005; 154 (5):1031-1044
8. Patricia Kunda,Gavin Craig,Veronica Dominguez,et al.Abi,Sral,and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions.Curr Biol 2005;13:1867-1875
9. Fan PD,Goff SP.Abl interactor 1 binds to sos and inhibits epidermal growth factor and v-Abl-induced activation of extracellular signal-regulated kinases.Mol Cell Biol 2005;20(20):7591-7601
10. Courtney KD,Grove M,Vandongen H,Vandongen A,et al.Localization and phosphorylation of Abl-interactor proteins,Abi1 and Abi-2,in the developing nevous system.Mol Cell Neurosci 2005;16(3):244-257