中枢神经再生抑制因子及免疫治疗研究

成体哺乳动物中枢神经损伤后早期轴突再生失败的一个主要原因是由于髓磷脂抑制分子的存在。Nogo、髓磷脂相关糖蛋白以及少突胶质细胞髓磷脂糖蛋白等神经再生抑制因子的发现,大大促进了中枢神经再生分子机制的研究。它们均能独立通过Nogo-66受体产生对轴突再生的抑制效应,髓磷脂抑制分子及其信号转导机制的研究日益成为中枢神经再生的研究热点,髓磷脂及其信号转导分子特别是Nogo-66受体、p75神经营养素受体成为损伤后促进轴突再生、抑制生长锥塌陷的主要治疗靶点。抑制上述抑制因子及相关受体NgR或p75NTR可能有助于中枢神经损伤的修复,围绕这些抑制因子及其相关受体介导的信号转导途径,人们提出了多种治疗中枢神经损伤的新思路,其中免疫学方法尤其受到关注。     

髓鞘相关糖蛋白与神经系统的髓鞘发育和轴突生长

髓鞘相关糖蛋白（myelin-associated glycoprotein,MAG）是免疫球蛋白超家族成员,它由中枢神经系统的少突胶质细胞和外周神经系统的施万细胞表达。MAG定位于直接和轴突相接触的髓鞘膜的最里层,它通过介导胶质细胞与轴突的相互作用参与髓鞘的形成及其完整性的维持。同时MAG也是髓鞘来源的神经生长抑制因子的主要成分。在神经系统发育的不同阶段,MAG显示不同的功能：即发育期促进轴突生长,成熟期抑制轴突生长。其抑制作用主要由髓鞘来源的抑制分子的共同受体NgR介导,在神经营养因子受体p75NTR以及小GTP酶Rho等信号分子的共同参与下完成。     

平滑肌细胞骨架结构及其信号调节途径

平滑肌细胞骨架是一个复杂的动态性网络,是细胞生命活动不可缺少的细胞结构。Rho通过活化其下游靶分子促进应力纤维的形成,其中Rho—associated coiled—coil kinase(ROCK)和Dial在该过程中起关键作用;PKC通过在细胞内不同定位的亚型使细胞骨架蛋白磷酸化,发挥其调节细胞骨架重构的作用。两条信号转导途径通过Src途径相互联系,共同参与细胞骨架动力学的调节。     

Rho GTPases及其在口腔领域的新应用

Rho GTPases是重要的信号转导分子,参与多种重要的细胞生命活动,如肌动蛋白细胞骨架的重构、细胞黏附、细胞运动、囊泡运输、基因表达和细胞周期的调控等.调节这些生物信号的转导通路非常复杂,因此,Rho GTPases早已成为研究的热点.最新的研究进展集中在描述Rho GTPases具体在细胞的哪个部位发生反应与参与通路的具体的分子及新功能等,同时细胞分子实验已经证实Rho GTPases在肌动蛋白细胞骨架的组装、细胞粘附、细胞运动、和基因表达等方面的作用与临床上多种口腔疾病,如正畸,牙周疾病的发生有密切关系.因此,对Rho GTPases的研究可能为口腔领域正畸,牙周病的治疗提供新的思路.     

Nogo A和srGAP蛋白在NIH 3T3细胞中的共表达

为检测Nogo A和srGAPs蛋白在NIH 3T3细胞上的表达,应用Western印迹的方法检测Nogo A蛋白的表达. 从NIH 3T3细胞抽提物中检测到约230 kD特异性的Nogo A反应条带;利用双重免疫细胞荧光化学标记法和激光共聚焦显微镜成像技术分别检测Nogo A与srGAPs或Rho蛋白在NIH 3T3细胞上的共表达状况,可观察到Nogo A与srGAPs共存于3T3细胞的细胞浆、突起和生长锥样结构上,亦可观察到Nogo A与Rho蛋白的共存.结果表明,NIH 3T3细胞中共表达Nogo A、srGAPs和Rho分子. 这为研究Nogo A与Rho信号转导途径间的关系奠定了基础.     

Rho小G蛋白介导的细胞周期调控

Rho小G蛋白作为一个信号分子家族具有多样化的功能, 可以调节细胞骨架重排 、细胞迁移、细胞极性、基因表达、细胞周期调控等. Rho小G蛋白家族对细胞周期 调控的研究主要集中在其对于有丝分裂期细胞的调节作用,包括调节有丝分裂期前 期细胞趋圆化、后期染色体排列及收缩环的收缩作用.近期的研究显示,Rho小G蛋白及其效应分子对于细胞周期G1、S、G2期的调控主要是通过影响细胞周期的正调控因子细胞周期蛋白D1 (cyclin D1) 和负调控因子细胞周期蛋白依赖型激酶相互作用蛋白1及细胞周期蛋白依赖型激酶抑制蛋白27 (p21cip1/p27kip1) 进行的.本文总结了Rho小G蛋白及其效应分子在细胞周期调控,尤其是对G1/S期调控的研究进展,并简要阐述了Rho小G蛋白介导的细胞周期调控异常与癌症发生的关系.     

硫酸软骨素蛋白多糖与神经系统的发育和再生

硫酸软骨素蛋白多糖（chondroitin sulfate proteoglycans,CSPGs）是中枢神经系统（CNS）细胞外基质中的重要组成成分,在CNS的发育、成熟后正常功能的维持中发挥重要功能,如发育中影响神经细胞的迁移和轴突生长,成年后参与神经可塑性的控制等;而病理条件下,如CNS受损后又可做为胶质瘢痕的重要组分抑制受损神经的再生。研究发现,用酶降解CSPGs的糖氨多糖链或阻断其合成可以有效地削弱CSPGs对受损神经的抑制作用,促进轴突再生。然而,精确调控CSPGs特定时空表达模式的分子机制,以及功能发挥所涉及的完整信号转导通路还有待进一步研究。     

脊髓损伤再生轴突生长的数学模型和三维格子波尔兹曼法数值模拟

中枢神经系统损伤是当今社会最具破坏力的疾病之一,虽然已经有办法使损伤后残存的神经元出芽,但如何保证处于萌芽状态的再生轴突继续生长直至与远端的靶细胞正确连接,是困扰至今的难题。为探讨中枢神经损伤所形成的胶质瘢痕和其所诱导的抑制因子对再生轴突生长进程的影响,根据轴突生长速度与其微环境中影响因子的浓度梯度成比例的原理,以脊髓损伤为背景构建数学模型,并采用格子波尔兹曼法进行三维数值模拟。数值试验中的主要观察指标为:1)当微环境中轴突生长抑制因子释放率和促进因子释放率一定时,胶质瘢痕的轴向厚度对轴突生长速率的影响,并跟踪记录生长锥所经过路线上的抑制因子浓度和促进因子浓度;2)当胶质瘢痕的轴向厚度一定时,抑制因子释放率和促进因子释放率对轴突生长速率的影响,并跟踪记录生长锥所经过路线上的抑制因子浓度和促进因子浓度。结果表明:1)胶质瘢痕的轴向厚度越大、抑制因子的释放率越强,轴突生长速率越小;2)轴突生长速率本质上取决于生长锥所在位置抑制因子浓度与促进因子浓度的比值,当该比值平均小于某个阈值时,再生轴突能够顺利生长并与靶细胞成功对接。为正确设计有关动物试验提供了理论参考。     

Rho激酶抑制剂在神经退化性疾病上的应用

Rho激酶,又称Rho相关的卷曲蛋白激酶,是一类丝氨酸/苏氨酸蛋白激酶,被发现为小G蛋白Rho的下游作用底物。由于Rho激酶活性涉及神经细胞的功能,而且越来越多的研究表明抑制Rho激酶的活性在数种神经退行性疾病包括帕金森病、阿尔茨海默病、亨廷顿病、多发性硬化症,和肌萎缩性侧索硬化症等的实验模式中都有明显的效果。因此,Rho激酶已成为针对治疗神经性退化性疾病的一个热门标靶蛋白。本文探讨Rho激酶抑制剂在神经退化性疾病上的应用及发展,使神经退行性疾病能进一步提升治疗和在应用上的水平。     

Rho GTPases和细胞凋亡

细胞凋亡涉及细胞骨架的形态学改变,Rho GTPases在细胞骨架改变中起着至关重要的作用。近年来的研究揭示了Rho蛋白家族在肌动蛋白(actin)聚合、解聚及actin-myosin的分子调节机制。同时越来越多的研究表明,Rho GTPases在巨噬细胞吞噬凋亡小体中也发挥了关键作用。本综述就Rho GTPases信号途径在细胞凋亡中细胞骨架的结构改变及凋亡小体被吞噬过程中的作用进行具体讨论。     

The suppression of small GTPase rho signal transduction pathway inhibits angiogenesis in vitro and in vivo

Angiogenesis consists of multistep pathways such as the degradation of the matrix, proliferation of the endothelial cells, motility of the endothelial cells, formation of the cord structure and network formation of microvessels. The small GTPase Rho participates in cell motility through actin fiber polymerization. The role of the small GTPase Rho signal transduction pathway in regulating angiogenesis, however, is still unknown. In this study, we investigated the role of the small GTPase Rho signal transduction pathway in angiogenesis in vitro and in vivo using the exoenzyme, Clostridium botulinum C3 transferase, which specifically suppresses Rho and a compound, Y-27632, which suppresses p160ROCK (Rho-associated coiled-coil containing protein kinase). In this paper, we showed that the small GTPase Rho-p160ROCK signal transduction pathway played an important role in angiogenesis both in vitro and in vivo. These results suggest that inhibition of the small GTPase Rho signal transduction pathway by the p160ROCK inhibitor could be a possible new strategy for angiogenic diseases.     

Rho kinase promotes alloimmune responses by regulating the proliferation and structure of T cells

Coordinated rearrangements of the actin-myosin cytoskeleton facilitate early and late events in T cell activation and signal transduction. As many important features of cell shape rearrangement involve small GTP-binding proteins, we examined the contribution of Rho kinase to the functions of mature T cells. Inhibitors of the Rho kinase pathway all had similar actions to inhibit the proliferation of primary lymphocyte cultures. Likewise, transfection of the human Jurkat T cell line with a dominant negative, kinase-defective mutant of Rho kinase diminished Jurkat cell proliferation. Furthermore, inhibition of Rho kinase substantially attenuated the program of cytokine gene expression that characterizes T cell activation, blocked actomyosin polymerization, and prevented aggregation of the TCR/CD3 complex colocalized with lipid rafts. These actions are relevant to immune responses in vivo, as treatment with a Rho kinase inhibitor considerably prolonged the survival of fully allogeneic heart transplants in mice and diminished intragraft expression of cytokine mRNAs. Thus, Rho GTPases acting through Rho kinase play a unique role in T cell activation during cellular immune responses by promoting structural rearrangements that are critical for T cell signaling.     

Small Rho GTPases are important for acinus formation in a human salivary gland cell line

Rho GTPases participate in a wide variety of signal transduction pathways regulating the actin cytoskeleton, gene expression, cellular migration and proliferation. The aim of this study was to evaluate the role of Rho GTPases in signal transduction pathways during acinus formation in a human salivary gland (HSG) cell line initiated by extracellular matrix (ECM; Matrigel) alone or in combination with epidermal growth factor, basic fibroblast growth factor and lysophosphatidic acid (LPA). Immunohistochemical and Western blotting analyses showed that HSG cells contained RhoA, RhoB, Rac1 and Cdc42 proteins. All growth factors enhanced the effects of ECM on acinus formation, in a pathway dependent on PI3-kinase and Rho GTPases. The role of ROCK, a major RhoA effector, seemed limited to cortical actin polymerization. LPA stimulated cell migration and acinus formation in a PI3-kinase-independent pathway. The results suggest that Rho proteins are important for epithelial-mesenchymal interactions during salivary gland development.This work was supported by FAPESP (grant numbers: 97/09507-6, 01/09047-2).     

Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1.

Wnt signaling via the Frizzled (Fz) receptor controls cell polarity and movement during development, but the molecular nature of Wnt/Fz polarity signal transduction remains poorly defined. Here we report that in human cells and during Xenopus embryogenesis, Wnt/Fz signaling activates the small GTPase Rho, a key regulator of cytoskeleton architecture. Wnt/Fz activation of Rho requires the cytoplasmic protein Dishevelled (Dvl) and a novel Formin homology protein Daam1. Daam1 binds to both Dvl and Rho, and mediates Wnt-induced Dvl-Rho complex formation. Inhibition or depletion of Daam1 prevents Wnt/Fz activation of Rho and of Xenopus gastrulation, but not of beta-catenin signaling. Our study illustrates a molecular pathway from Wnt/Fz signaling to Rho activation in cell polarity signal transduction.     

Genetic analysis demonstrates a direct link between rho signaling and nonmuscle myosin function during Drosophila morphogenesis

A dynamic actomyosin cytoskeleton drives many morphogenetic events. Conventional nonmuscle myosin-II (myosin) is a key chemomechanical motor that drives contraction of the actin cytoskeleton. We have explored the regulation of myosin activity by performing genetic screens to identify gene products that collaborate with myosin during Drosophila morphogenesis. Specifically, we screened for second-site noncomplementors of a mutation in the zipper gene that encodes the nonmuscle myosin-II heavy chain. We determined that a single missense mutation in the zipper(Ebr) allele gives rise to its sensitivity to second-site noncomplementation. We then identify the Rho signal transduction pathway as necessary for proper myosin function. First we show that a lethal P-element insertion interacts genetically with zipper. Subsequently we show that this second-site noncomplementing mutation disrupts the RhoGEF2 locus. Next, we show that two EMS-induced mutations, previously shown to interact genetically with zipper(Ebr), disrupt the RhoA locus. Further, we have identified their molecular lesions and determined that disruption of the carboxyl-terminal CaaX box gives rise to their mutant phenotype. Finally, we show that RhoA mutations themselves can be utilized in genetic screens. Biochemical and cell culture analyses suggest that Rho signal transduction regulates the activity of myosin. Our studies provide direct genetic proof of the biological relevance of regulation of myosin by Rho signal transduction in an intact metazoan.     

Modulation of epithelial tubule formation by Rho kinase

We have developed a model system for studying integrin regulation of mammalian epithelial tubule formation. Application of collagen gel overlays to Madin-Darby canine kidney (MDCK) cells induced coordinated disassembly of junctional complexes that was accompanied by lamellipodia formation and cell rearrangement (termed epithelial remodeling). In this study, we present evidence that the Rho signal transduction pathway regulates epithelial remodeling and tubule formation. Incubation of MDCK cells with collagen gel overlays facilitated formation of migrating lamellipodia with membrane-associated actin. Inhibitors of myosin II and actin prevented lamellipodia formation, which suggests that actomyosin function was involved in regulation of epithelial remodeling. To determine this, changes in myosin II distribution, function, and phosphorylation were studied during epithelial tubule biogenesis. Myosin II colocalized with actin at the leading edge of lamellipodia thereby providing evidence that myosin is important in epithelial remodeling. This possibility is supported by observations that inhibition of Rho kinase, a regulator of myosin II function, alters formation of lamellipodia and results in attenuated epithelial tubule development. These data and those demonstrating myosin regulatory light-chain phosphorylation at the leading edge of lamellipodia strongly suggest that Rho kinase and myosin II are important modulators of epithelial remodeling. They support a hypothesis that the Rho signal transduction pathway plays a significant role in regulation of epithelial tubule formation. signaling pathway; polarity     

Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase

The ephrins, ligands of Eph receptor tyrosine kinases, have been shown to act as repulsive guidance molecules and to induce collapse of neuronal growth cones. For the first time, we show that the ephrin-A5 collapse is mediated by activation of the small GTPase Rho and its downstream effector Rho kinase. In ephrin-A5-treated retinal ganglion cell cultures, Rho was activated and Rac was downregulated. Pretreatment of ganglion cell axons with C3-transferase, a specific inhibitor of the Rho GTPase, or with Y-27632, a specific inhibitor of the Rho kinase, strongly reduced the collapse rate of retinal growth cones. These results suggest that activation of Rho and its downstream effector Rho kinase are important elements of the ephrin-A5 signal transduction pathway.     

Rho kinase activation maintains high pulmonary vascular resistance in the ovine fetal lung

Mechanisms that maintain high pulmonary vascular resistance (PVR) in the fetal lung are poorly understood. Activation of the Rho kinase signal transduction pathway, which promotes actin-myosin interaction in vascular smooth muscle cells, is increased in the pulmonary circulation of adult animals with experimental pulmonary hypertension. However, the role of Rho kinase has not been studied in the fetal lung. We hypothesized that activation of Rho kinase contributes to elevated PVR in the fetus. To address this hypothesis, we studied the pulmonary hemodynamic effects of brief (10 min) intrapulmonary infusions of two specific Rho kinase inhibitors, Y-27632 (15-500 microg) and HA-1077 (500 microg), in chronically prepared late-gestation fetal lambs (n = 9). Y-27632 caused potent, dose-dependent pulmonary vasodilation, lowering PVR from 0.67 +/- 0.18 to 0.16 +/- 0.02 mmHg x ml(-1) x min(-1) (P < 0.01) at the highest dose tested without lowering systemic arterial pressure. Despite brief infusions, Y-27632-induced pulmonary vasodilation was sustained for 50 min. HA-1077 caused a similar fall in PVR, from 0.39 +/- 0.03 to 0.19 +/- 0.03 (P < 0.05). To study nitric oxide (NO)-Rho kinase interactions in the fetal lung, we tested the effect of Rho kinase inhibition on pulmonary vasoconstriction caused by inhibition of endogenous NO production with nitro-L-arginine (L-NA; 15-30 mg), a selective NO synthase antagonist. L-NA increased PVR by 127 +/- 73% above baseline under control conditions, but this vasoconstrictor response was completely prevented by treatment with Y-27632 (P < 0.05). We conclude that the Rho kinase signal transduction pathway maintains high PVR in the normal fetal lung and that activation of the Rho kinase pathway mediates pulmonary vasoconstriction after NO synthase inhibition. We speculate that Rho kinase plays an essential role in the normal fetal pulmonary circulation and that Rho kinase inhibitors may provide novel therapy for neonatal pulmonary hypertension.