Tenascin-C在周围神经再生过程中的功能和相关分子机制研究

发布时间：2018-08-20 08:44

【摘要】：周围神经损伤是一种常见的临床疾病。周围神经系统在损伤后是具有一定再生能力的,而且周围神经再生是一个涉及许多细胞活动的复杂的生物学过程,其损伤及再生的确切机制尚未完全阐明。有大量研究表明,雪旺细胞在周围神经再生的过程中扮演了极其重要的角色,也是造成中枢神经系统与周围神经系统再生能力迥异的一个重要原因。在周围神经系统受到损伤后,损伤位置附近的雪旺细胞会经历脱髓鞘的过程,并去分化为类似发育过程中未成熟状态的雪旺细胞,在这个状态下,雪旺细胞又会再次进入细胞周期开始分裂增殖,并沿着再生的轴突向前迁移,最后包绕新生的轴突再次形成髓鞘,以达到修复神经的目地。然而,目前关于神经损伤后再生的相关的调节机制仍不完全清楚,而且该过程由于涉及到多种细胞类型以及大量的基因,因此为了找到在周围神经再生过程中的一些关键调控基因并进一步阐明相关的核心基因在神经再生中的作用,我们便借助了生物信息学的方法,以期发现并研究损伤后再生过程中重要核心调控因子的功能,并为临床药物的研发和组织工程神经的构建提供基础。在本研究中,我们首先通过外科手术制作了大鼠坐骨神经离断模型,通过表达谱芯片技术分析坐骨神经损伤后0,1,4,7和14天坐骨神经近侧残端5 mm的神经组织,并采用随机方差模型对各个时间点的样本进行差异基因及表达趋势的显著性分析,我们共检测到6046个差异基因(P0.01, FDR0.05),经KEGG分析,获得关键信号通路及对整个信号通路基因表达变化影响最显著的核心调控基因,结果显示tenascin-C (TNC)在神经损伤再生这一过程中处于调控网络的核心地位,暗示了TNC在神经再生过程中可能扮演着重要的角色。TNC是一种胞外基质大分子蛋白,是tenascin家族最早被发现的成员,往往被发现在发育过程中和损伤部位表达丰富,而在成年个体的组织中表达较少。此外,TNC还被发现在肿瘤系统中有着促进多种肿瘤细胞迁移和侵袭的能力,并且在中枢神经系统的相关研究中,TNC还被发现具有促进轴突生长的能力,然而在周围神经系统中目前关于TNC的研究还很少,并且结合TNC在前文生物信息学分析中显示出的重要作用,在周围神经系统中对TNC进行深入的研究是有着很大的必要性的。在后续的深入研究中,我们发现了在坐骨神经遭受到横断损伤后,坐骨神经中近侧断端的TNC会在损伤4天后持续地维持在很高的表达水平,而在正常神经组织中的表达却很低,进一步的暗示了TNC在周围神经再生过程中的重要作用。之后我们又通过了免疫荧光的方法分别检测了TNC与雪旺细胞标志物S100β,巨噬细胞标志物CD68和成纤维细胞标志物P4HB(prolyl-4-hydroxylase beta)的共定位情况,发现了在周围神经损伤后TNC几乎不与雪旺细胞和巨噬细胞共定位,而与成纤维细胞则共定位良好,说明了成纤维细胞在坐骨神经损伤后会大量表达TNC。由于各种创伤均会造成不同程度的细胞变性、坏死和组织缺损,必须通过细胞增殖和细胞间基质的形成来进行组织修复。在此修复过程中,成纤维细胞起着十分重要的作用。以伤口愈合过程为例,成纤维细胞通过有丝分裂大量增殖,并合成和分泌大量的胶原纤维和基质成分,参与损伤后组织的修复与重建。Alison Lloyd等人在2010年发现了在周围神经损伤后,成纤维细胞会聚集到损伤部位,通过EphB信号引导雪旺细胞轴向排列,以促进周围神经的再生。基于这篇报道以及我们发现的神经损伤后成纤维细胞会大量分泌TNC这一现象,我们提出了成纤维细胞在周围神经受到损伤后会大量表达TNC以影响雪旺细胞的某些生物学过程这个假设。为了验证这一假设,我们首先在体外设计了一个基于transwell的成纤维细胞-雪旺细胞共培养系统,我们发现了在transwell共培养系统的下层种入的成纤维细胞的数量会影响迁移过transwell小室的雪旺细胞的数量,二者是呈正比关系的,证明了成纤维细胞确实具有影响雪旺细胞迁移的能力,并且证明了这一共培养系统的有效性。之后为了验证TNC是否是成纤维细胞影响雪旺细胞的一个媒介,我们设计了TNC的siRNA,并在RNA水平和蛋白水平验证了siRNA的基因沉默效率,并分别通过了transwell实验和细胞划痕实验证明了抑制成纤维细胞TNC的表达可以显著降低与之共培养的雪旺细胞的迁移能力；之后我们又通过使用外源性的TNC处理单独体外培养的雪旺细胞,发现了在培养液中加入1μ g/ml和10μ g/ml的TNC蛋白可以有效地促进雪旺细胞的迁移,结合上面的结果,说明了TNC是成纤维细胞影响雪旺细胞迁移的一个重要媒介。之后,我们还通过体内实验发现,相比对照组,外源性的1μg/ml和10μg/ml TNC蛋白可以在坐骨神经损伤后显著促进雪旺细胞的迁移速度和迁移数量,而且对于轴突的生长也有有效的促进作用。除了考查TNC对于雪旺细胞迁移的影响之外,我们还考察了TNC对于雪旺细胞的另外两个重要功能,即增殖和成髓鞘的影响,通过体外细胞培养实验我们发现在培养液中加入1μ g/ml和10μ g/ml的TNC蛋白后,雪旺细胞的增殖速度与对照组没有出现显著差异,说明了TNC对于雪旺细胞的增殖没有影响；之后为了考查TNC对于雪旺细胞成髓鞘能力的影响,我们使用了体外雪旺细胞-神经元的共培养成髓鞘模型,我们发现在诱导成髓鞘的过程中加入1 μ g/ml的TNC蛋白对于雪旺细胞的成髓鞘过程会出现明显的抑制。整联蛋白是一种跨膜的异质二聚体,它由α和β两个非共价结合的跨膜亚基。细胞外球形结构域是一个露出细胞膜外约20 nm的头部,头部可同细胞外基质蛋白结合。整联蛋白的两个亚基,α和β链都是糖基化的,并通过非共价键结合在一起。整联蛋白是一种介导细胞和其外环境(主要是细胞外基质)之间的连接的跨膜受体。在信号转导中,整联蛋白将细胞外基质的化学成分与力学状态等有关信息传入细胞并且参与了细胞通信、细胞周期以及细胞运动等多项细胞功能的调节。在周围神经损伤和再生的过程中,雪旺细胞发挥了非常重要的作用,而整联蛋白也被发现具有影响着雪旺细胞的多种生物学行为(包括迁移,增殖及成髓鞘)的能力。我们前期的芯片数据分析预测了整联蛋白β 1很可能作为TNC的下游基因在再生过程中起着关键的作用。因此为了研究TNC与整联蛋白β1是否存在着上下游的关系,我们首先设计了整联蛋白β1的siRNA并分别在mRNA水平和蛋白水平验证了其基因沉默效率,之后我们发现在使用siRNA干扰体外培养雪旺细胞的整联蛋白β1后,TNC促进雪旺细胞的迁移的效果消失了；除此之外,我们还直接在蛋白水平使用抗体封闭了雪旺细胞表面的整联蛋白β1,与siRNA干扰的结果相似,在封闭了整联蛋白β 1的功能后,TNC促进雪旺细胞的迁移的效果也消失了；而之后为了更为直观的研究TNC是否能直接的与雪旺细胞表面的整联蛋白β1相结合,我们又使用了一个免疫共沉淀实验显示了TNC与整联蛋白β 1的直接结合,综合上面的结果,我们可以得出TNC是通过结合到雪旺细胞表面的整联蛋白β1进而影响雪旺细胞的迁移这一结论。接下来,我们检测了整联蛋白β1下游与迁移相关的Rho GTPase家族的几个重要效应分子,发现了TNC可以诱导RhoA和Racl而非Cdc42的激活,且对其总表达量无影响。而之后的实验也发现Racl的抑制剂而非RhoA的抑制剂可以显著降低由TNC诱导的雪旺细胞迁移,进一步确定了TNC是通过结合雪旺细胞表面受体整联蛋白β1,进一步使下游的Racl激活,以促进雪旺细胞的迁移。综合以上结果表明,TNC在坐骨神经再生过程中是成纤维细胞调节施万细胞迁移的一个重要调节因子,是二者存在密切相互作用的一个新证据。成纤维细胞相对于坐骨神经的其它组成细胞(如雪旺细胞,神经元,巨噬细胞等)目前还缺乏相对系统的研究。而近几年,已经有越来越多的从事周围神经再生领域研究的学者将研究的注意力放到成纤维细胞上,尤以Alison Lloyd等人在2010年首次对于成纤维细胞和雪旺细胞的相互作用做出了深入的研究,让我们对于周围神经系统中成纤维细胞的功能有了一个全新的认识。随着对于周围神经成纤维细胞研究的继续深入,成纤维细胞在周围神经再生过程中发挥的作用将会越来越多的被探索出来,极可能是未来周围神经再生领域研究的一个重要方向。
[Abstract]:Peripheral nerve injury is a common clinical disease. Peripheral nerve regeneration is a complex biological process involving many cellular activities. The exact mechanism of injury and regeneration of peripheral nerve has not been fully elucidated. Schwann cells in the vicinity of the injured peripheral nervous system undergo demyelination and dedifferentiate into Schwann fines similar to those in the immature state of development. In this state, Schwann cells enter the cell cycle again and begin to divide and proliferate, migrate forward along the regenerated axon, and finally form myelin sheath around the regenerated axon to repair the nerve. In order to find some key regulatory genes involved in peripheral nerve regeneration and further elucidate the role of related core genes in nerve regeneration, we have resorted to bioinformatics methods in order to discover and study the important core modulations in the process of regeneration after injury. In this study, we first established a rat model of sciatic nerve amputation by surgical operation, and then analyzed the 5 mm nerve tissue of the proximal sciatic nerve stump at 0, 1, 4, 7 and 14 days after sciatic nerve injury by expression spectrum chip technique. Random variance model was used to analyze the significance of different genes and their expression trend. We detected 6046 different genes (P 0.01, FDR 0.05). KEGG analysis showed that tenascin-C (TNC) had the most significant effect on the change of gene expression in the whole signal pathway. Nerve regeneration is at the core of the regulatory network, suggesting that TNC may play an important role in nerve regeneration. In addition, TNC has been found to promote the migration and invasion of a variety of tumor cells in the tumor system. In related studies of the central nervous system, TNC has also been found to promote axonal growth. However, there are few studies on TNC in the peripheral nervous system, and TNC binding precedes it. In subsequent in-depth studies, we found that TNC in the proximal sciatic nerve was maintained at a very high level 4 days after sciatic nerve transection. After that, we detected TNC and Schwann cell marker S100beta, macrophage marker CD68 and fibroblast marker P4HB (prolyl-4-hydroxylase b) by immunofluorescence. The co-localization of ETA showed that TNC almost did not co-localize with Schwann cells and macrophages after peripheral nerve injury, but co-localized well with fibroblasts, suggesting that fibroblasts would express TNC in large quantities after sciatic nerve injury. Fibroblasts play an important role in tissue repair by cell proliferation and intercellular matrix formation. In wound healing, for example, fibroblasts proliferate through mitosis, and synthesize and secrete a large number of collagen fibers and matrix components, which participate in tissue repair and repair after injury. Alison Lloyd et al. found in 2010 that after peripheral nerve injury, fibroblasts aggregated to the injured site and directed Schwann cells to arrange axially through EphB signals to promote peripheral nerve regeneration. Based on this report and our findings, fibroblasts secrete a large amount of TNC after nerve injury, we propose that To verify this hypothesis, we first designed a transwell-based fibroblast-Schwann cell co-culture system in vitro. We found that the underlying layer of the Transwell co-culture system The number of fibroblasts seeded into the cells affected the number of Schwann cells that migrated through the Transwell chamber, which was in direct proportion to the number of fibroblasts. This proved that fibroblasts did have the ability to affect the migration of Schwann cells and the effectiveness of the co-culture system. We designed siRNA for TNC as a mediator, and validated the gene silencing efficiency of siRNA at RNA and protein levels. Transwell and cell scratch experiments showed that inhibition of TNC expression in fibroblasts significantly reduced the migration ability of Schwann cells co-cultured with TNC; and then we made it possible to inhibit the expression of TNC in fibroblasts. In vitro cultured Schwann cells were treated with exogenous TNC. It was found that the addition of 1 and 10 ug/ml TNC proteins in the culture medium could effectively promote the migration of Schwann cells. Combined with the above results, TNC was an important mediator of fibroblasts affecting the migration of Schwann cells. Compared with the control group, exogenous 1 ug/ml and 10 ug/ml TNC protein could significantly promote the migration rate and quantity of Schwann cells after sciatic nerve injury, and also promote the growth of axons. In addition to examining the effect of TNC on the migration of Schwann cells, we also investigated the effect of TNC on Schwann cells. Two other important functions, proliferation and myelination, we found that the proliferation rate of Schwann cells was not significantly different from that of the control group after adding 1 and 10 ug/ml TNC protein in the culture medium, which indicated that TNC had no effect on the proliferation of Schwann cells. We used the Schwann cell-neuron co-culture myelination model in vitro to investigate the effect of 1 ug/ml TNC protein on the myelination of Schwann cells. Two non-covalently bound transmembrane subunits of beta. An extracellular spherical domain is a head that exposes an extracellular membrane about 20 nm and binds to extracellular matrix proteins. Both subunits of the integrin, alpha and beta chains, are glycosylated and bind through non-covalent bonds. The integrin is a mediator of cells and their external environment (mainly In signal transduction, integrins transmit information about the chemical composition and mechanical state of the extracellular matrix into cells and participate in the regulation of cellular communication, cell cycle and cell movement. In the process of peripheral nerve injury and regeneration, Schwann is fine. Cells play a very important role, and integrins have been found to influence a variety of biological behaviors of Schwann cells, including migration, proliferation and myelination. Our previous data analysis on chip data predicted that integrin beta 1 may play a key role in the regeneration process as a downstream gene of TNC. To investigate the relationship between TNC and integrin beta 1, we first designed siRNA of integrin beta 1 and validated its gene silencing efficiency at mRNA and protein levels, respectively. Then we found that TNC promoted the migration of Schwann cells by interfering with integrin beta 1 of Schwann cells cultured in vitro with siRNA. In addition, we blocked integrin beta 1 on the surface of Schwann cells directly at the protein level with antibodies, similar to the results of siRNA interference. After blocking the function of integrin beta 1, the effect of TNC on promoting Schwann cell migration disappeared; and then, for a more intuitive study of whether TNC can directly interact with Schwann cells. Combining the above results, we can conclude that TNC affects the migration of Schwann cells by binding to integrin beta 1 on the surface of Schwann cells. Several important effector molecules of the Rho GTPase family associated with migration downstream of integrin beta 1 were measured and TNC was found to induce the activation of RhoA and Racl rather than Cdc42 without affecting the total expression of RhoA. TNC is further activated by binding to Schwann cell surface receptor integrin beta 1 to promote the migration of Schwann cells. These results suggest that TNC is an important regulator of Schwann cell migration by fibroblasts during sciatic nerve regeneration, and there is a close interaction between TNC and Schwann cell migration. There is a new evidence that fibroblasts are not systematically studied in relation to other components of the sciatic nerve (such as Schwann cells, neurons, macrophages, etc.). In recent years, more and more researchers in the field of peripheral nerve regeneration have focused their attention on fibroblasts, especially Alison Lloyd. The first in-depth study of the interaction between fibroblasts and Schwann cells in 2010 has led to a new understanding of the function of fibroblasts in the peripheral nervous system. The role will be more and more explored, which is likely to be an important direction in the field of peripheral nerve regeneration in the future.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R741

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