蝾螈肢体再生中终末分化细胞去分化调控机制的研究进展

两栖类有尾目动物蝾螈具有极强的再生能力,尤其在肢体受损被截断后能在较短时间内达到复原,而哺乳动物不具备肢体再生的能力。在肢体再生中,残肢处的终末分化细胞发生去分化是诱导再生芽基形成,启动断肢再生的重要始动步骤。现从蝾螈断肢再生的组织形态、细胞生物学、信号分子和神经免疫调控等方面对终末分化细胞去分化调控机制的进展进行综述,为肢体再生医学的研究提供借鉴。     

肢体再生——来自有尾两栖类的认知

蝾螈等有尾两栖类在其肢体任何节段被截断后,能通过准确的时空模式调节完成具有位置匹配关系的再生修复,该过程由受损肢体残端产生的芽基组织介导完成。芽基细胞的来源目前尚有争议,其产生受局部基质微环境诱导并涉及细胞表观遗传学改变,性状上呈现不完全的细胞再编程特征,增殖分化具有神经依赖性。哺乳类包括人类仅具有极为有限的肢体再生能力,其肢体再生限于指（趾）末端受损离断。深入探讨有尾两栖类等肢体再生过程的细胞分子机制,将为探索新的干细胞损伤修复途径及再生促进策略提供线索。     

编委推荐

正Developmental Cell|成纤维细胞去分化是断肢再生成功的决定因素脊椎动物中只有两栖动物能够再生断肢。有尾两栖动物蝾螈在一生各阶段都可以再生断肢,而无尾两栖动物爪蛙在变态后只有很有限的断肢再生能力。爪蛙断肢能够形成再生芽基,但其再生仅限于形成棘状软骨,而且不分节,即没有图式建成。是什么因素导致爪蛙断肢再生发生了缺陷?爪蛙断肢芽基的细胞,与蝾螈芽基细胞究竟有什么不同?     

论交提要

1.利用单位和三价染色体移植物研究表皮在两棲类肢体再生中的作用The role of epithelium in amphibian limbregeneration,studied by haploid and triploidtransplants,E.D.Hav,Am.J.Anat.91:447—481,1952.本实验的目的是验证 Rose 关于两棲类肢体再生过程中去分化表皮细胞参与早期再生芽基形成的学说、豹蛙(Rana pipiens)元单价、双价和三价染色体的蝌蚪,按不同的配合方式分别用作施主和寄主。将施主之尾部表皮移植到寄主蝌蚪右后肢的大腿部位,左后肢留作对照。然后,通过两肢体的膝关节处切     

无神经蝾螈幼虫前肢摘除肱骨后的再生

1.年青的无神经幼虫前肢,切断并摘除肱骨后可以再生,除去在少数例子肱骨的再生不完全外,大多数的再生都是典型的。再生体形态建成速度也和同年龄的正常幼虫相近。2.再生芽基细胞起源于残肢中仅有的肌肉和结缔组织,其中肌肉在数量上比结缔组织多,去分化过程也非常明显,因此,作为早期再生芽基细胞的来源,肌肉组织可能比结缔组织更为重要。3.由肌肉和结缔组织去分化而来的芽基细胞,不仅能够分化为肌肉和结缔组织,而且能分化出典型的软骨。这表明芽基细胞的多潜能性,由某种组织去分化而来的细胞可以分化为另一种组织。4.表皮细胞在再生初期表现出形态去分化特征,它和内部组织之间有密切的连系,但是没有看到表皮细胞直接参入内部。     

赤子爱胜蚓再生过程中肌细胞超微结构变化的研究

将赤子爱胜蚓在60／61体节处剪切,用透射电镜观察肌肉再生过程中的形态变化。结果表明：剪切后伤口处的受损细胞呈降解状态并被迁移至伤口面的吞噬细胞所吞噬;伤口处的其他未受损细胞去分化,失去特化细胞的特征,并且增殖形成圆锥状的芽基;芽基细胞再次进行分化,成肌细胞合成肌丝,最终生成新的肌肉组织。     

不同年龄蝾螈肢体再生速度的比较

1.根据外形与组织学的观察,比较了不同年龄的东方蝾螈(Cynops orientalis)肢体的再生速度。我们发现再生速度随年龄增加而降低。幼虫的再生速度最高,变态以后再生速度显著地下降,但幼体(一岁、尚未达性成熟)仍比成体(性成熟)快得多。2.幼虫肢体再生之一切组织学过程均比变态后的动物进行快。幼体和成体的前二再生时期(组织去分化期和再生芽基形成期)进行速度相差不大,它们之间的差别主要在于分化期中再生体之生长和分化速度。3.再生初期速度(即组织去分化速度)依赖于断切时肢体组织的分化程度。变态前后组织分化程度的巨大差异造成了幼虫和幼体、成体之间组织去分速度的巨大差别。幼虫肢体组织分化程度低,去分化比变态后进行快得多;幼体和成体肢体所有组织的分化均接近或已达到完成,形态上彼此没有显著差异,这可能解释为什么它们的去分速度都减慢而且彼此大约相等。4.实验期中幼体体长仍继续增加,而成体生长几已陷于停顿,这可能是它们的再生体生长速度的差别的原因。此外,也应考虑到性成熟过程中组织生理的变化可能对再生体的生长和分化发生影响。5.我们讨论了内分泌和再生的关系并认为变态和性成熟过程中内分泌变化所引起的组织生理和组织分化程度的差...     

神经系统对断肢再生的调控

两栖动物蝾螈和爪蛙是断肢再生能力最强的脊椎动物,其断肢再生的一个鲜明的特点是对神经组织的成瘾性依赖。关于断肢再生神经依赖的研究已有近两百年的历史,但神经与再生芽基和顶外胚层帽交流互作的机制仍不明朗。该文以蝾螈与爪蛙断肢再生为例,简要回顾神经支配断肢再生的研究结果,并结合作者实验室最近有关黑皮质素受体信号通路调控爪蛙蝌蚪断肢再生的研究进展,探讨中枢神经系统对断肢再生的调控机理。     

昆虫肢体再生的研究进展

文中就发生断肢再生的昆虫类群、出现虫态、再生的类型、再生能力、影响再生的因素、再生的生物学意义几个方面进行了综述 ,特别对猎蝽科昆虫的肢体再生有关方面做了介绍     

植物再生——自然界的神秘力量

<正>自然界的植物再生现象动物的器官再生现象广为人知,如壁虎在遇到危险时可以采取断尾来摆脱险境并再生出新的尾部,蝾螈也可以断肢自救并随后再生出新的肢体。同样的,自然界的植物也可以利用再生来应对外界生长环境的变化:多肉植物的离体叶片可以在伤口处生出新芽和新根,并最终形成新植株;白杨的枝条通过扦插可以长出不定     

金鱼再生的视神经在顶盖投射精确化的DiI顺行标记研究

本文用微量显微注射法,在金鱼视网膜的背侧用亲脂类荧光染料DiI标记少量神经节细胞,通过顺行标记研究了视神经再生过程中视网膜顶盖投射的精确化过程。在损伤视神经后的不同时期观察了再生视神经纤维在顶盖整装片上的分布。在再生早期它们以超出正常的途径由背腹两侧进入顶盖,广泛分布。但其中大部分仍分布于顶盖腹侧的靶区。在再生晚期通过精确化,重建如正常鱼一样精确的视网膜顶盖投射。这个精确化过程表现在以下三方面:(1)再生于顶盖错误区域的再生视神经纤维的消失;(2)再生早期视神经纤维主干上生长的侧部分支的消失;(3)到达靶区的再生视神经纤维形成重迭的终末分支。由以上结果推测,顶盖中可能存在两类不同的因子:一类是普通诱向因子,存在于整个顶盖中,它在再生早期引导再生的视神经纤维长入顶盖。另一类是神经营养因子,它具区域特异性,在再生晚期引导视神经纤维到达顶盖靶区,形成精确的视网膜顶盖投射。     

A comparison of the neurotrophic activities of the flavonoid fisetin and some of its derivatives

Neurotrophic factors promote the development, maintenance and regeneration of nerve cells. Classical neurotrophic factors are proteins and thus not well-suited for therapeutic purposes. Recently, we showed that specific flavonoids such as fisetin (3, 7, 3', 4' tetrahydroxyflavone) promote the differentiation of nerve cells in culture through the activation of extracellular signal-regulated kinase (ERK) suggesting that flavonoids could substitute for neurotrophic factors. It has also been shown that fisetin promotes nerve cell survival following exposure to toxic oxidative insults. To determine whether or not this is unique to fisetin, a series of related compounds were assayed for neurotrophic activities. Many of these related compounds also promote nerve cell differentiation and are neuroprotective against toxic oxidative insults. However, the mechanisms underlying these neurotrophic effects differ among the compounds.     

Urokinase plasminogen receptor and the fibrinolytic complex play a role in nerve repair after nerve crush in mice, and in human neuropathies

Remodeling of extracellular matrix (ECM) is a critical step in peripheral nerve regeneration. In fact, in human neuropathies, endoneurial ECM enriched in fibrin and vitronectin associates with poor regeneration and worse clinical prognosis. Accordingly in animal models, modification of the fibrinolytic complex activity has profound effects on nerve regeneration: high fibrinolytic activity and low levels of fibrin correlate with better nerve regeneration. The urokinase plasminogen receptor (uPAR) is a major component of the fibrinolytic complex, and binding to urokinase plasminogen activator (uPA) promotes fibrinolysis and cell movement. uPAR is expressed in peripheral nerves, however, little is known on its potential function on nerve development and regeneration. Thus, we investigated uPAR null mice and observed that uPAR is dispensable for nerve development, whereas, loss of uPAR affects nerve regeneration. uPAR null mice showed reduced nerve repair after sciatic nerve crush. This was a consequence of reduced fibrinolytic activity and increased deposition of endoneurial fibrin and vitronectin. Exogenous fibrinolysis in uPAR null mice rescued nerve repair after sciatic nerve crush. Finally, we measured the fibrinolytic activity in sural nerve biopsies from patients with peripheral neuropathies. We showed that neuropathies with defective regeneration had reduced fibrinolytic activity. On the contrary, neuropathies with signs of active regeneration displayed higher fibrinolytic activity. Overall, our results suggest that enforced fibrinolysis may facilitate regeneration and outcome of peripheral neuropathies.     

Molecular approaches to nerve regeneration.

Current research into regeneration of the nervous system has focused on defining the molecular events that occur during regeneration. One well-characterized system for studying nerve regeneration is the sciatic nerve of rat. Numerous studies have characterized the sequence of events that occur after a crush injury to the sciatic nerve (Cajal 1928; Hall 1989). These events include axon and myelin breakdown, changes in the permeability of the blood vessels, proliferation of Schwann cells, invasion of macrophages, and the phagocytosis of myelin fragments by Schwann cells and macrophages. The distal segment of the injured sciatic nerve provides a supportive environment for the regeneration of the nerve fibres (Cajal 1928; David & Aguayo 1981). Within a period of weeks, the injured sciatic nerve is able to regrow and successfully reinnervate the appropriate targets. Some of the molecules that provide trophic support for the regrowing nerve fibres have been identified, including nerve growth factor (NGF) (Heumann et al. 1987) and glial maturation factor beta (Bosch et al. 1989). Another class of molecules show changes in their rates of synthesis during regeneration, including both proteins (Skene & Shooter 1983; Muller et al. 1986) and mRNA species (Trapp et al. 1988; Meier et al. 1989). To better understand nerve regeneration, we have taken two, parallel molecular approaches to study the events associated with regeneration. The first of these is to study in detail the mechanism of action of a molecule that has been implicated in the regeneration process, nerve growth factor. The second approach is to identify novel gene sequences which are regulated during regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of thyroid hormones and their receptors in peripheral nerve regeneration.

After peripheral nerve injury in adult mammals, reestablishment of functional connections depends on several parameters including neurotrophic factors, the extracellular matrix, and hormones. However, little is known about the contribution of hormones to peripheral nerve regeneration. Thyroid hormones, which are required for the development and maturation of the central nervous system, are also important for the development of peripheral nerves. The action of triiodothyronine (T3) on responsive cells is mediated through nuclear thyroid hormone receptors (TRs) which modulate the expression of specific genes in target cells. Thus, to study the effect of T3, it is first necessary to know whether the target tissues possess TRs. The fact that sciatic nerve cells possess functional TRs suggests that these cells can respond to T3 and, as a consequence, that thyroid hormone may be involved in peripheral nerve regeneration. The silicone nerve guide model provides an excellent system to study the action of local administration of T3. Evidence from such studies demonstrate that animals treated locally with T3 at the level of transection have more complete regeneration of sciatic nerve and better functional recovery. Among the possible regulatory mechanisms by which T3 enhances peripheral nerve regeneration is rapid action on both axotomized neurons and Schwann cells which, in turn, produce a lasting and stimulatory effect on peripheral nerve regeneration. It is probable that T3 up- or down-regulates gene expression of one or more growth factors, extracellular matrix, or cell adhesion molecules, all of which stimulate peripheral nerve regeneration. This could explain the greater effect of T3 on nerve regeneration compared with the effect of any one growth factor or adhesion molecule.     

Acidic fibroblast growth factor enhances peripheral nerve regeneration in vivo

When added to a collagen-filled nerve guide, purified acidic fibroblast growth factor (aFGF) increased the number of myelinated axons that regenerated across a 5-mm nerve gap distance. In addition, a greater number of primary sensory and motor neurons extended axons through the nerve guide in animals treated with aFGF. Thus the effect of aFGF on peripheral nerve regeneration is not simply an increase in axonal branching within the nerve guide tube. This is the first highly purified growth factor since nerve growth factor that has been shown to promote nerve regeneration in vivo. This experimental model provides a convenient and quantitative means to assess the effects of putative neuronotropic factors on peripheral nerve regeneration in vivo.     

Extracellular vesicles from human umbilical cord mesenchymal stem cells improve nerve regeneration after sciatic nerve transection in rats

Peripheral nerve injury results in limited nerve regeneration and severe functional impairment. Mesenchymal stem cells (MSCs) are a remarkable tool for peripheral nerve regeneration. The involvement of human umbilical cord MSC‐derived extracellular vesicles (hUCMSC‐EVs) in peripheral nerve regeneration, however, remains unknown. In this study, we evaluated functional recovery and nerve regeneration in rats that received hUCMSC‐EV treatment after nerve transection. We observed that hUCMSC‐EV treatment promoted the recovery of motor function and the regeneration of axons; increased the sciatic functional index; resulted in the generation of numerous axons and of several Schwann cells that surrounded individual axons; and attenuated the atrophy of the gastrocnemius muscle. hUCMSC‐EVs aggregated to rat nerve defects, down‐regulated interleukin (IL)‐6 and IL‐1β, up‐regulated IL‐10 and modulated inflammation in the injured nerve. These effects likely contributed to the promotion of nerve regeneration. Our findings indicate that hUCMSC‐EVs can improve functional recovery and nerve regeneration by providing a favourable microenvironment for nerve regeneration. Thus, hUCMSC‐EVs have considerable potential for application in the treatment of peripheral nerve injury.     

Increased muscle regeneration after repair of divided motor nerve with neuronotrophic factors containing glue

Neuronotrophic factors (NTFs) directed to spinal cord motor neurons were collected in rats within silicone nerve regeneration chambers according to LONGO et al. (1983b). Unilateral addition of NTFs to the fibrin glue used for the repair of divided sciatic nerves improved locally nerve regeneration without affecting the controlateral side. Nerve regeneration was assessed by weight gain of the reinnervated muscles and by radioactive labelling of the acid-soluble phosphate fractions of both nerve Schwann cells and reinnervated muscle cells. Fast gastrocnemius and slow soleus muscles, the motor nerve of which had been repaired with added NTFs, were significantly heavier (21 and 28%) than their controlateral controls, and the metabolic dedifferentiation attendant on post-division nerve repair was less marked. It is suggested that this experimental nerve regeneration model is suitable to test potential nerve-active agents in vivo, under conditions close to the usual clinical setting, with, as ultimate goal, the improvement of the end-results of microsurgical repair of peripheral nerve in man.     

Effect of a high-intensity static magnetic field on sciatic nerve regeneration in the rat

The effect of a high-intensity static magnetic field on peripheral nerve regeneration is evaluated in rat sciatic nerve. Forty-four rats underwent sciatic nerve repair using polyethylene nerve guides. Postoperatively, the animals were exposed to a 1-tesla magnetic field for 12 hours per day for 4 weeks with appropriate controls. Our results demonstrate that a 1-tesla static magnetic field has no statistically significant effect on nerve regeneration as determined by myelinated axon counts and electrophysiologic studies. Also, the specific orientation of the sciatic nerve with respect to the magnetic field has no influence on axonal growth or nerve conduction. Periods of restraint of 12 hours per day for 4 weeks significantly inhibit weight gain but have no effect on peripheral nerve regeneration.