【作者】 向强；

【导师】 周跃；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【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。更多还原

【Abstract】 BackgroundThe 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 Materials1. 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 myelolysisThe 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 expression1) 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.Results1. 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 myelolysisThere 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 expressionThere 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 sequencing2) 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.Conclusion1. 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.更多还原