新生大鼠半横切脊髓内胚胎脊髓移植后大脑皮层cDNA差示文库构建及部分相关基因分析
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摘要
愈来愈多的研究表明，提供适当条件后损伤的CNS能够再生，轴突的再生是由神经元固有的特性和其所处的环境暗示(environmental cues)共同决定的。 影响CNS再生的因素很多，主要分为内在因素和外在因素两个方面，内在因素是指与CNS神经元轴突中断有关的基因表达，外在因素是指CNS中抑制轴突再生的分子和/或物理屏障。影响轴突生长外在因素也能通过诱导内在的神经基因的表达变化而发挥作用。实验证明直接给予损伤神经元胞体营养因子处理可逆转细胞萎缩，增强生长相关蛋白-43(GAP-43)和Tα-1微管蛋白(Tα-1 tubulin) mRNA表达，促进轴突再生。内在因素方面，CNS神经元在轴突中断后基因表达在数量及种类上与PNS明显不同，寻找并增强再生相关基因中关键基因的表达,以促进CNS损伤后轴突再生和/或出芽的反应是神经科学研究的重要方向。
研究表明，新生大鼠CNS的再生能力强于成年大鼠，而应用胚胎脊髓移植处理脊髓损伤后的新生大鼠,与未处理的新生大鼠有更强的再生能力。提示，接受胚胎脊髓移植后，可能刺激新生大鼠的神经元表达功能更有意义的再生相关基因。为了探索胚胎脊髓移植后新生大鼠再生过程中皮层神经元基因表达的变化，探讨相关的再生机制及寻找更有效的治疗方法，本课题采用新生大鼠脊髓半横切模型，并即刻局部移植胚胎脊髓，结合SMART技术和SSH方法构建再生相关基因差异显示文库，并对验证为阳性的EST片段进行测序、克隆及同源性分析，另外,对一条可能与再生相关的新基因进行电子克隆、初步验证及生物信息学分析，初步证明，其在CNS轴突再生中可能有重要作用。
主要研究结果和结论如下：
1．本实验建立了新生1-2天大鼠双侧脊髓半横切及单侧胚胎脊髓移植的模型，该模型能客观反映皮质脊髓束对肢体运动功能单侧和交叉支配的结构特点，易于功能观察，致伤因素单一，致伤条件一致,可实现同一动物体内双侧对比，是一种稳定、重复性良好的再生研究模型。
2．用原位杂交的方法观察到，胚胎脊髓移植后5天损伤脊髓GAP-43mRNA强
烈表达，提示选择该时间窗观察再生相关基因表达是可行的。同时，与未损伤组及未移植组相比，移植组GAP-43mRNA的表达显著增强，表明我们建立的实验模型能够满足实验的需要。
3．用SMART和SSH相结合的办法构建了新生大鼠脊髓损伤及胚胎脊髓移植后5天皮层神经元cDNA差异显示文库。分析文库中的基因，发现新生大鼠脊髓损伤胚胎脊髓移植后第5天，基因表达变化范围广泛，包括与细胞能量和蛋白合成代谢、细胞增殖活动、促进细胞迁移及细胞生长的诱导调控、细胞生物氧化和磷酸化作用等有关基因。表明脊髓损伤后再生过程由ERK/MAPK(mitogen-activated protein kinase, MAPK) 通路等多种信号转导系统参与，并涉及多种生物化学作用。
4．我们在GenBank中成功注册了25个新EST序列。
5．用Northern blot 检测H3(CA854305)变化，实验显示新生大鼠脊髓损伤后5天，给予胚胎脊髓移植组对侧大脑皮层中的H3表达，与未移植组和未损伤组有显著性差异，移植组强于未移植组，未移植组强于未损伤组；提示H3可能与再生关系密切。分析该基因定位于大鼠第8号染色体q31。电子克隆获得最大长度1635bp,最大开放阅读框位于49-591bp,结构分析符合Cozak规则。
6．用RT-PCR试验初步验证了最大开放阅读框的序列，对推测翻译的蛋白序列在Genbank中进行BLASTP,证明与人类N-乙酰氨基葡萄糖-6-0-磺基转移酶具有高度相似性(98％)。分析该蛋白一级、二级和三级结构，并结合该蛋白结构上存在的蛋白酶作用位点，我们认为 H3基因及其推导蛋白质分子可能是分布于CNS中特殊部位并与CNS低分化状态有一定关系的信号分子。
Numerous studies now show that anatomical regeration and functional recovery are possible if given proper conditions in injuried CNS, and the axon regeneration is decided by both the instinct properties of neurons and the environment cues abround them. It is becoming increasingly evident that several factors are implicated in the failure of central nervous system neurons to regenerate their axons after injury，these factors are often conceptualized as being either intrinsic or extrinsic.The instrinc factors is pointed to the factors that related to the native gene expression response to the axotomized CNS neuron. The extrinsic ones are ,for example, the factors that related to molecules and/or physical barriers in the CNS environment that inhibit axonal growth in nature. In fact, extrinsic factors such as molecules that influence growth cone motility/ guidance may also induce intrinsic factors such as neuronal gene expression. It was proved that cell body treatment of axotomized neurons with neurotrophins reversed atrophy, increased GAP-43 and Tα-1 tubulin mRNA expression, and promoted axonal regeneration into peripheral nerve grafts. With regarding to the extrinsic factors, the gene expression variety in CNS are qualitatively and/or quantitatively different from that in PNS. Seeking for the key genes associating with regeneration and strengthening their expression in CNS to promote the axonal regeneration and/or neuritis sprouting is an very essential direction in neuroscience research.
It has been proved that the CNS of newborn rats have more strengthened regeneration ability compared with the adult rats, and the newborn injured rats accepted fetal spinal cord transplantation have more strengthened regeneration ability compared with the newborn rats without fetal spinal cord transplantation. It is suggested the newborn rats accepted the fetal spinal cord transplantation may express novel genes associating with regeneration that maybe have more stronger regeneration ability. To explore the variational gene expression in cerebral cortical neurons of newborn rats during their stage of regeneration after injury, to grope for the related regeneration mechanism ,and to ask for more efficiently therapeutic methods, our experiments
prepared the spinal cord hemisection model, transplanted the fetal spinal cord immediately after the injury at the local injuried position, constructed the differential expression cDNA library combining the methods of SMART and SSH, analysised the positive clones and BLASTN the EST sequences, in silico cloned one gene from the library and BLASTP the putatively protein, also we considered that the protein may play important roles in axonal regeneration of CNS.
The main results and conclusions are as below:
1. We constructed the spinal cord hemisection model in newborn 1-2 days rats, transplanted fetal spinal cord into the local injuried place immediately. The model can impersonally reflect the unilateral and cross characteristic in structure and function of CST, it is easy investigated and repeated, with identical causative factors in same environment, it is an ideal model for the study of regeneration after spinal cord injury .
2. Using in situ hybridization methods, we observed that the injuried spinal cord express GAP-43 mRNA extensively 5 days after fetal spinal cord transplantation, it implied that the time point of 5 days after injury is proper. Otherwise, the transplantation group express strengthened GAP-43 efficiently compared with the untreated and uninjuried groups, so we concluded that the model we constructed is perfect for our experiment.
3. Combining the techniques of SMART with SSH, we constructed the differential expression cDNA library of the cortical neurons of newborn rats 5 days after the spinal cord injury and fetal spinal cord transplantation. After BLASTNing the genes we screened from the library, we found the genes on the highest identities with them are involved in many biological processings, including synthesis of energy and protein, cell prolifer
研究表明，新生大鼠CNS的再生能力强于成年大鼠，而应用胚胎脊髓移植处理脊髓损伤后的新生大鼠,与未处理的新生大鼠有更强的再生能力。提示，接受胚胎脊髓移植后，可能刺激新生大鼠的神经元表达功能更有意义的再生相关基因。为了探索胚胎脊髓移植后新生大鼠再生过程中皮层神经元基因表达的变化，探讨相关的再生机制及寻找更有效的治疗方法，本课题采用新生大鼠脊髓半横切模型，并即刻局部移植胚胎脊髓，结合SMART技术和SSH方法构建再生相关基因差异显示文库，并对验证为阳性的EST片段进行测序、克隆及同源性分析，另外,对一条可能与再生相关的新基因进行电子克隆、初步验证及生物信息学分析，初步证明，其在CNS轴突再生中可能有重要作用。
主要研究结果和结论如下：
1．本实验建立了新生1-2天大鼠双侧脊髓半横切及单侧胚胎脊髓移植的模型，该模型能客观反映皮质脊髓束对肢体运动功能单侧和交叉支配的结构特点，易于功能观察，致伤因素单一，致伤条件一致,可实现同一动物体内双侧对比，是一种稳定、重复性良好的再生研究模型。
2．用原位杂交的方法观察到，胚胎脊髓移植后5天损伤脊髓GAP-43mRNA强
烈表达，提示选择该时间窗观察再生相关基因表达是可行的。同时，与未损伤组及未移植组相比，移植组GAP-43mRNA的表达显著增强，表明我们建立的实验模型能够满足实验的需要。
3．用SMART和SSH相结合的办法构建了新生大鼠脊髓损伤及胚胎脊髓移植后5天皮层神经元cDNA差异显示文库。分析文库中的基因，发现新生大鼠脊髓损伤胚胎脊髓移植后第5天，基因表达变化范围广泛，包括与细胞能量和蛋白合成代谢、细胞增殖活动、促进细胞迁移及细胞生长的诱导调控、细胞生物氧化和磷酸化作用等有关基因。表明脊髓损伤后再生过程由ERK/MAPK(mitogen-activated protein kinase, MAPK) 通路等多种信号转导系统参与，并涉及多种生物化学作用。
4．我们在GenBank中成功注册了25个新EST序列。
5．用Northern blot 检测H3(CA854305)变化，实验显示新生大鼠脊髓损伤后5天，给予胚胎脊髓移植组对侧大脑皮层中的H3表达，与未移植组和未损伤组有显著性差异，移植组强于未移植组，未移植组强于未损伤组；提示H3可能与再生关系密切。分析该基因定位于大鼠第8号染色体q31。电子克隆获得最大长度1635bp,最大开放阅读框位于49-591bp,结构分析符合Cozak规则。
6．用RT-PCR试验初步验证了最大开放阅读框的序列，对推测翻译的蛋白序列在Genbank中进行BLASTP,证明与人类N-乙酰氨基葡萄糖-6-0-磺基转移酶具有高度相似性(98％)。分析该蛋白一级、二级和三级结构，并结合该蛋白结构上存在的蛋白酶作用位点，我们认为 H3基因及其推导蛋白质分子可能是分布于CNS中特殊部位并与CNS低分化状态有一定关系的信号分子。
Numerous studies now show that anatomical regeration and functional recovery are possible if given proper conditions in injuried CNS, and the axon regeneration is decided by both the instinct properties of neurons and the environment cues abround them. It is becoming increasingly evident that several factors are implicated in the failure of central nervous system neurons to regenerate their axons after injury，these factors are often conceptualized as being either intrinsic or extrinsic.The instrinc factors is pointed to the factors that related to the native gene expression response to the axotomized CNS neuron. The extrinsic ones are ,for example, the factors that related to molecules and/or physical barriers in the CNS environment that inhibit axonal growth in nature. In fact, extrinsic factors such as molecules that influence growth cone motility/ guidance may also induce intrinsic factors such as neuronal gene expression. It was proved that cell body treatment of axotomized neurons with neurotrophins reversed atrophy, increased GAP-43 and Tα-1 tubulin mRNA expression, and promoted axonal regeneration into peripheral nerve grafts. With regarding to the extrinsic factors, the gene expression variety in CNS are qualitatively and/or quantitatively different from that in PNS. Seeking for the key genes associating with regeneration and strengthening their expression in CNS to promote the axonal regeneration and/or neuritis sprouting is an very essential direction in neuroscience research.
It has been proved that the CNS of newborn rats have more strengthened regeneration ability compared with the adult rats, and the newborn injured rats accepted fetal spinal cord transplantation have more strengthened regeneration ability compared with the newborn rats without fetal spinal cord transplantation. It is suggested the newborn rats accepted the fetal spinal cord transplantation may express novel genes associating with regeneration that maybe have more stronger regeneration ability. To explore the variational gene expression in cerebral cortical neurons of newborn rats during their stage of regeneration after injury, to grope for the related regeneration mechanism ,and to ask for more efficiently therapeutic methods, our experiments
prepared the spinal cord hemisection model, transplanted the fetal spinal cord immediately after the injury at the local injuried position, constructed the differential expression cDNA library combining the methods of SMART and SSH, analysised the positive clones and BLASTN the EST sequences, in silico cloned one gene from the library and BLASTP the putatively protein, also we considered that the protein may play important roles in axonal regeneration of CNS.
The main results and conclusions are as below:
1. We constructed the spinal cord hemisection model in newborn 1-2 days rats, transplanted fetal spinal cord into the local injuried place immediately. The model can impersonally reflect the unilateral and cross characteristic in structure and function of CST, it is easy investigated and repeated, with identical causative factors in same environment, it is an ideal model for the study of regeneration after spinal cord injury .
2. Using in situ hybridization methods, we observed that the injuried spinal cord express GAP-43 mRNA extensively 5 days after fetal spinal cord transplantation, it implied that the time point of 5 days after injury is proper. Otherwise, the transplantation group express strengthened GAP-43 efficiently compared with the untreated and uninjuried groups, so we concluded that the model we constructed is perfect for our experiment.
3. Combining the techniques of SMART with SSH, we constructed the differential expression cDNA library of the cortical neurons of newborn rats 5 days after the spinal cord injury and fetal spinal cord transplantation. After BLASTNing the genes we screened from the library, we found the genes on the highest identities with them are involved in many biological processings, including synthesis of energy and protein, cell prolifer
引文
1. Mckerracher L. Spinal cord repair: Strategies to promote axon regeneration. Neurobiology of disease,2001,(8):11-18
2. Richardson PM, Issa VM, Aguayo AJ. Regeneration of long spinal axons in the rat. J Neurocytol, 1984,13:165-182.
3. Schreyer DJ, Skene JH. Injury-associated induction of GAP-43 expression displays axon branch specificity in rat dorsal root ganglion neurons. J Neurobiol, 1993,24:959-970.
4. Broude E, McAtee M, Kelley MS ，et al. c-Jun expression in adult rat dorsal root ganglion neurons: differential response after central or peripheral axotomy. Exp Neurol, 1997, 148:367-377.
5. Neumann S, Woolf CJ. Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury. Neuron,1999, 23:83-91.
6. Berry M, Carlile J, Hunter A. PN explants grafted into the vitreous body of the eye promote the regeneration of retinal ganglion cell axons severed in the optic nerve. J Neurocytol, 1996, 25:147-170.
7. Leon S, Yin Y, Nguyen J, et al. Lens injury stimulates axon regeneration in the mature rat optic nerve. J Neurosci, 2000, 20:4615-4626.
8. Bregman BS and Bernstein-Goral H. Both regenerating and late-developing pathways contribute to transplant-induced anatomical plasticity after spinal cord lesions at birth. Experimental neurology,1991,112:49-63
2. Richardson PM, Issa VM, Aguayo AJ. Regeneration of long spinal axons in the rat. J Neurocytol, 1984,13:165-182.
3. Schreyer DJ, Skene JH. Injury-associated induction of GAP-43 expression displays axon branch specificity in rat dorsal root ganglion neurons. J Neurobiol, 1993,24:959-970.
4. Broude E, McAtee M, Kelley MS ，et al. c-Jun expression in adult rat dorsal root ganglion neurons: differential response after central or peripheral axotomy. Exp Neurol, 1997, 148:367-377.
5. Neumann S, Woolf CJ. Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury. Neuron,1999, 23:83-91.
6. Berry M, Carlile J, Hunter A. PN explants grafted into the vitreous body of the eye promote the regeneration of retinal ganglion cell axons severed in the optic nerve. J Neurocytol, 1996, 25:147-170.
7. Leon S, Yin Y, Nguyen J, et al. Lens injury stimulates axon regeneration in the mature rat optic nerve. J Neurosci, 2000, 20:4615-4626.
8. Bregman BS and Bernstein-Goral H. Both regenerating and late-developing pathways contribute to transplant-induced anatomical plasticity after spinal cord lesions at birth. Experimental neurology,1991,112:49-63