Rho激酶通路对大鼠海马神经元突起生长和微管重排的调节

发布时间：2018-06-28 14:21

本文选题：Rho激酶 + 微管　；参考：《南方医科大学》2007年博士论文

【摘要】： 目的和意义: 神经系统是由数千亿神经元通过突触连接形成的一个既精确又复杂的神经网络。神经元的发育过程中,经历了从分化、迁移、极化、定向生长到与靶细胞建立突触联系的动态过程。其突起末端的生长锥感受细胞外生长和导向信息,通过微管和微丝运动使细胞形成树突和轴突,并不断生长、延伸或坍塌。细胞骨架系统不断进行重排是细胞突起生长、确定方向和迁移的关键。Rho家族的鸟苷三磷酸酶(Rho GTPases)是调节细胞骨架运动的主要信号,细胞外生长和导向因子首先通过细胞膜上相应的受体启动Rho激酶,进而通过坍塌反应调节蛋白(CRMPs)影响细胞骨架的重排,如微管和微丝的聚合和解聚,而细胞骨架重排的结果是神经元突起的生长、延伸或坍塌。神经系统发育过程中赋予神经元复杂的结构及其它们形成的精确的神经网络构成了神经系统功能活动的基础,神经元的发育不仅是从幼小到成熟的过程,也涉及损伤后的修复和再生过程,神经组织损伤后突起的再生过程也被认为是其发育过程的再现。探讨Rho激酶通路对神经元突起生长和微管重排的调节不仅有助于理解神经损伤后的再生过程,也可以加深对神经退行性病变发生的认识,使我们有机会找到克服神经组织再生和修复困难的新思路和干预靶点。材料和方法: 实验首先以发育不同阶段的大鼠海马为对象,提取总RNA,用RT-PCR的方法检测大鼠海马发育过程中突起生长导向因子GAP-43、Nogo-A、netrin-1、Sema 3A和Nrp-1 mRNA,以及Rho通路信号分子Rho A、Rac-1、CRMP-1和Tubβ3 mRNA的表达水平。进而以原代大鼠海马神经元为对象,用Rho激酶促进剂LPA和抑制剂Y27632干预海马神经元,LPA和Y27632的浓度200ng/ml,原子力显微镜观察细胞突起的生长,并提取突起进行定量;共聚焦显微镜观察细胞微管的重排,并对粘附蛋白vinculin进行亚细胞定位和分布特征进行观察。实验进一步构建Rho激酶的下游信号CRMP-1的重组体,用非定向克隆技术构建CRMP-1-phrGFPⅡ-N真核表达载体,阳性脂质体法转染NGF诱导分化的PC12细胞,观察CRMP-1对细胞突起生长的影响。结果: (1)大鼠海马发育过程中生长导向因子和Rho通路信号分子mRNA的表达大鼠海马的发育过程中,生长因子GAP-43 mRNA胚胎和新生阶段表达较多,幼年和成年阶段表达较少(P＜0.05),老年阶段表达仍维持较高水平;Nogo-A在胚胎阶段表达较多,出生后和GAP-43相似,成年和老年阶段处于高峰时期。导向因子netrin-1以胎鼠、新生鼠和成年鼠表达最强,幼年和老年阶段表达较少。Sema 3A随着海马发育逐渐降低,但其受体Nrp-1从幼年到老年表达逐渐增多。信号分子Rho-A和Rac-1的表达与Rho-A的表达有相似的变化规律,随着海马发育亦呈明显的阶段性,表达的高峰在胚胎、幼年和老年阶段,而新生阶段和成年阶段处于相对低表达阶段;Rac-1的表达量在海马发育的各个阶段均超过Rho-A的表达量。CRMP-1和tubulin的表达从胚胎到成年的各个阶段与CRMP-1的表达具有相似的变化趋势,以胎鼠、新生大鼠和幼年大鼠表达较多,但老年阶段CRMP-1的表达较成年阶段显著升高(P＜0.05),而tubulin在老年阶段停留在成年阶段的水平。 (2)Rho激酶对大鼠海马神经元突起生长和微管重排的调节原子力显微镜的观察显示,LPA处理组海马神经元突起的数量减少,一些突起仅见残留的突起根部,一至三级突起的数目均减少(P＜0.05);LPA预处理后用Y27632干预,虽然胞体的形态未见明显的变化,但是一级突起的数目增多,呈辐射状从胞体伸出,并伴有丰富的二级突起,一级和二级突起的数目与对照组相比增多(P＜0.05);提取突起的光密度测定显示,LPA处理后的细胞较对照组降低(P＜0.05),LPA预处理后用Y27632干预组的细胞较LPA组升高(P＜0.05),与对照组无差异显著。共聚焦显微镜观察显示,海马神经元胞体、突起和生长锥均显示清晰的微管,LPA干预组突起较短,而且突起远端的荧光较浅淡;胞体内无清晰可见的丝网状微管,取而代之的是不规则的微管,其粗细不均,排列不规整,失去原有的鸟巢样外观;突起内微管的排列出现发辫样改变,成束的微管之间出现较大的缝隙;生长锥失去原有的外貌,仅见突起细小的末端,无典型的生长锥,其中的微管大部分消失。LPA预处理后用Y27632干预,胞体内可见较清晰的丝网状微管,这些微管粗细较均匀,除了围绕胞核周围分布的微管外,还可见末端游离的微管,伸入细胞的突起;突起内的微管不甚规整,仍可见微管之间较大的空隙,但较LPA组排列规整;突起末端生长锥基本保持了原有的形态,呈不典型的扇形突出,其中可见较清晰的微管,它们直行伸入生长锥或者弯曲走行,较长的微管伸入生长锥中心部位,短的微管位于生长锥的根部。vinculin蛋白散在分布于细胞膜上,LPA干预组细胞膜上亦可见绿色荧光点散在分布于细胞膜上;LPA处理后用Y27632干预,细胞膜上分布较密集的绿色荧光,而且这些荧光点常聚集在一起,在细胞膜的周边区域和突起的根部分布较密集,显示vinculin蛋白在细胞膜的周边聚集。 (3)CRMP-1-phrGFPⅡ-N真核表达载体的构建及其对突起生长的调节从大鼠海马总RNA扩增出的目的基因片断为1800bp,符合设计片断的大小;连接转化后形成白色菌落,EcoRⅠ单酶切后的重组体出现1800bp大小的目的基因片断和3000bp大小的克隆载体片断,未经酶切的重组体为4800bp。双向测序结果显示,CRMP-1目的基因片断为1746bp,与genbank的序列比对显示仅4位的碱基突变,不影响翻译蛋白质,同源性为99.8%。与表达载体连接后,转化细菌,挑取正向连接克隆,CRMP-1-phrGFPⅡ-N重组体电泳条带位于6700bp处,酶切后显示4900bp和1800bp两个片断。 50ng NGF诱导后,PCI2细胞突起延长,分化为神经元样细胞。空载体转染组的细胞显示较强的绿色荧光,荧光分布于胞体和突起,细胞的突起较长。CRMP-1-phrGFPⅡ-N转染组,其荧光与空载体转染组分布相似,主要分布于胞体和突起,但细胞突起较短小,表达CRMP-1的细胞少见超过胞体直径数倍的突起,个别较长的突起亦较空载体组短小。结论: (1)在大鼠海马的发育过程中,胚胎阶段GAP-43和Nogo-A处于高表达状态,出生后回落,老年阶段GAP-43仍维持较高水平,而Nogo-A在成年和老年均呈高表达状态,提示老年阶段海马神经元仍具有较强的生长和再生能力,而抑制因子的高表达可能是再生困难的原因。 (2)生长导向因子netrin-1的表达贯穿胚胎、新生、幼年和成年阶段,老年表达较低;而Sema 3A主要局限于胚胎和新生阶段,但是其受体的表达在经历了新生阶段的低表达后逐渐增多的过程。 (3)细胞内Rho-A和Rac-1信号分子的发育虽然在各个阶段均有较高的表达,但具有明显的阶段性,新生和成年阶段表达相对较低,胚胎、幼年和老年阶段相对较高。 (4)微管蛋白的发育从胚胎到幼年呈逐渐下降的趋势,成年和老年阶段相对稳定,CRMP-1的发育从胚胎到成年阶段与微管的发育经历相似的变化,但是老年阶段表达较高。 (5)激活Rho激酶通路可诱导神经元突起坍塌,表现在各级突起数目的减少和长度的缩短,抑制Rho激酶通路可促使神经元突起各级分支数目的增加以及突起的生长。Rho激酶不但通过调节微管重排参与突起的生长延长,还参与突起的分支形成,抑制Rho激酶的活性基本能逆转LPA对突起生长的坍塌作用,但并不能完全逆转LPA诱导的微管重排。 (6)扩增了CRMP-1的cDNA,完成CRMP-1-PGEM-T Easy克隆载体和CRMP-1-phrGFPⅡ-N真核表达载体的构建。 (7)转染由NGF诱导分化的PC12细胞后,CRMP-1在细胞的胞体和突起表达,导入CRMP-1外源基因的PC12细胞突起明显缩短,显示CRMP-1具有抑制突起生长和诱导其坍塌的作用。
[Abstract]:Purpose and significance:
A neural network is an accurate and complex neural network formed by a synapse of hundreds of billions of neurons through synapses. During the development of a neuron, a dynamic process from differentiation, migration, polarization, and directional growth to the target cells. Tube and microfilament movement causes cells to form dendrites and axons, growing, extending or collapsing. The cytoskeleton system constantly rearrangements is the growth of cell protuberances. The key.Rho family of.Rho (Rho GTPases) is the main signal to regulate cytoskeleton movement, and the extracellular growth and guiding factors are first passed. The corresponding receptor on the cell membrane starts the Rho kinase and then affects the rearrangement of the cytoskeleton through the collapse response regulating protein (CRMPs), such as the polymerization and disaggregation of microtubules and microfilaments, and the result of the rearrangement of the cytoskeleton is the growth, extension or collapse of the neurites. The complex structures and their shapes are given to the neurons in the process of the development of the nervous system. The exact neural network constitutes the basis of the functional activity of the nervous system. The development of neurons is not only from young to mature, but also in the process of repair and regeneration after injury. The process of regeneration of the neurites after the injury of the nerve tissue is also considered to be the reproduction of its development process. The growth of the neurites by the Rho kinase pathway and the growth of the neuron are discussed. The regulation of microtubule rearrangement not only helps to understand the process of regeneration after nerve injury, but also helps to deepen the understanding of the occurrence of neurodegenerative diseases, so that we have a chance to find new ideas and targets to overcome the difficulty of regeneration and repair of nerve tissue.
Materials and methods:
First, the total RNA was extracted from the hippocampus of rats at different stages of development. RT-PCR was used to detect the growth guiding factor GAP-43, Nogo-A, netrin-1, Sema 3A and Nrp-1 mRNA in the process of hippocampus development in rats, and the expression level of Rho pathway signal molecule Rho A. Rho kinase promoter LPA and inhibitor Y27632 were used to interfere with hippocampal neurons, LPA and Y27632 concentration 200ng/ml. The growth of cell protuberances was observed by atomic force microscopy, and the protuberances were extracted and quantified. Confocal microscopy was used to observe the rearrangement of cell microtubules and to observe subcellular localization and distribution characteristics of adherent egg white vinculin. The experiment further constructed the recombinant of the downstream signal CRMP-1 of the Rho kinase, and constructed the CRMP-1-phrGFP II -N eukaryotic expression vector by non directional cloning technique. The positive liposome method was transfected to NGF induced PC12 cells, and the effect of CRMP-1 on the growth of the cell protuberance was observed.
Result:
(1) expression of growth orientation factor and Rho pathway signaling molecule mRNA during hippocampus development in rats
During the development of rat hippocampus, the expression of growth factor GAP-43 mRNA embryo and new stage is more, the expression in young and adult stage is less (P < 0.05), the expression of old age is still high, Nogo-A is more expressed in the embryo stage, after birth and GAP-43, and at the peak period of adult and old age. The guide factor netrin-1 is born. The expression of rat, newborn rat and adult rat was the strongest. The expression of.Sema 3A in young and old age decreased gradually with the development of hippocampus, but the expression of its receptor Nrp-1 increased gradually from young to old. The expression of signal molecule Rho-A and Rac-1 was similar to the expression of Rho-A. With the development of hippocampus, the peak of expression was also obvious. In the embryo, the young and the old, the new stage and the adult stage are in the relatively low expression stage; the expression of Rac-1 is more than the expression of Rho-A,.CRMP-1 and tubulin at various stages of the development of the hippocampus. The expression of.CRMP-1 and the expression of the expression from the embryo to the adult is similar to the expression of the expression of the CRMP-1. There was more expression in rats, but the expression of CRMP-1 was significantly higher in the elderly stage than in the adult stage (P < 0.05), while tubulin remained at the adult stage in the elderly stage.
(2) Regulation of Rho kinase on neurite outgrowth and microtubule rearrangement in rat hippocampal neurons
Atomic force microscopy (AFM) showed that the number of neurites in the hippocampal neurons in the LPA treatment group decreased, some of the protuberances were only seen in the residual protuberance, and the number of the first to three protrusions decreased (P < 0.05). LPA was pretreated with Y27632, although the morphology of the cell body was not obviously changed, but the number of primary protuberances increased, showing radiation from the cell body. The number of the first and two stage protrusions increased (P < 0.05), the number of first and two protrusions was increased (P < 0.05). The cells after LPA treatment were lower than the control group (P < 0.05), and the cells in the Y27632 intervention group were higher than those in the LPA group (P < 0.05) after the pretreatment (P < 0.05), and there was no significant difference between the control group and the control group.
The confocal microscope showed that the cells of the hippocampal neurons, the protuberances and the growth cones all showed clear microtubules. The protuberances of the LPA intervention group were shorter, and the fluorescence of the protuberance was lighter. There was no clear visible microtubule in the cell, and the irregular microtubules were replaced with irregular arrangement and lost the original nests. The arrangement of microtubules in the protuberances appeared with braid changes and large gaps between the microtubules of the bundles; the growth cone lost the original appearance, only seen the fine ends and no typical growth cones. Most of the microtubules disappeared after the.LPA preconditioning, and Y27632 was used to intervene in the cells. More evenly, apart from the microtubules around the nucleus of the nucleus, the free microtubules of the end were visible, and the microtubules in the protuberances were not very regular, and the larger spaces between the microtubules were still visible, but they were arranged regularly in the LPA group. The clear microtubules are straight into the growth cone or bend and walk, the longer microtubules reach the central part of the growth cone, the short microtubules are scattered on the cell membrane at the root of the growth cone, and the green fluorescence points are scattered on the cell membrane on the cell membrane of the LPA intervention group; after LPA treatment, the cell membrane is interfered with the cell membrane. The dense green fluorescence is denser, and these fluorescent dots are often gathered together, denser in the surrounding area of the cell membrane and in the root of the protuberance, indicating that the vinculin protein is gathered around the cell membrane.
(3) construction of CRMP-1-phrGFP II -N eukaryotic expression vector and regulation of neurite outgrowth
The target gene fragment amplified from the total RNA of the rat hippocampus was 1800bp, conforming to the size of the design fragment. After the connection was transformed to form a white colony, the 1800bp size target gene fragment and the 3000bp size cloned carrier fragment appeared in the recombinant EcoR I after the single enzyme cut, and the unenzymed recombinant body was shown to 4800bp. bi-directional sequencing results, and CRMP-1 orders were shown. The gene fragment was 1746bp, and the sequence alignment with GenBank showed that only 4 base mutations did not affect the translation of protein. After the homology was connected to the expression vector, the homology was converted to bacteria, and the positive clones were picked up. The CRMP-1-phrGFP II -N recombinant strip was located at 6700bp, and the two fragments of 4900bp and 1800bp were displayed after the enzyme digestion.
After the induction of 50NG NGF, the protuberances of PCI2 cells were extended and differentiated into neuron like cells. The cells in the transfected group showed strong green fluorescence, and the fluorescence was distributed in the cell and the protuberance. The cell protuberance was long.CRMP-1-phrGFP II -N transfection group. The fluorescence of the cells was similar to the distribution of the empty body transfected group, mainly in the cell body and the protuberance, but the cell protruded more. In short, the cells expressing CRMP-1 rarely exceed the diameter of the cell body several times, and some longer protuberances are shorter than empty vectors.
Conclusion:
(1) during the development of hippocampus, the GAP-43 and Nogo-A in the embryonic stage were in high expression state, after birth, the GAP-43 remained high in the aged, while Nogo-A was highly expressed in both adult and old age, suggesting that the hippocampal neurons still have strong growth and regeneration ability in the aged and the high expression of inhibitory factors may be expressed. It's the reason why it's difficult to regenerate.
(2) the expression of growth guiding factor netrin-1 runs through the embryo, newborn, young and adult stage, and the expression of old age is low; and Sema 3A is mainly confined to the embryo and the newborn stage, but the expression of its receptor is gradually increasing after the low expression of the new stage.
(3) the development of Rho-A and Rac-1 signal molecules in cells has high expression at all stages, but it has obvious stages. The expression of new and adult stage is relatively low, and the embryo, young and old age are relatively high.
(4) the development of microtubulin is gradually decreasing from embryo to young age, and the development of adult and old age is relatively stable. The development of CRMP-1 is similar to the development of microtubules from embryo to adult stage, but the expression of old age is higher.
(5) activation of the Rho kinase pathway can induce the collapse of the neurite protuberance, the decrease of the number of protuberances at all levels and the shortening of the length. The inhibition of the Rho kinase pathway can promote the increase of the number of branches at all levels and the growth of the protuberance.Rho kinase not only by regulating the growth of the microtubule rearrangement and the protuberance, but also in the branching branching. Inhibition of Rho kinase activity can basically reverse the collapse of LPA to neurite growth, but it does not completely reverse LPA induced microtubule rearrangement.
(6) amplify the cDNA of CRMP-1, and complete the construction of CRMP-1-PGEM-T Easy cloning vector and CRMP-1-phrGFP II -N eukaryotic expression vector.
(7) after transfection of PC12 cells induced by NGF, CRMP-1 was expressed in cell body and protuberance, and the protuberances of PC12 cells introduced into CRMP-1 were obviously shortened, which showed that CRMP-1 had the effect of inhibiting the growth of protruding and inducing its collapse.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R338

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