神经营养因子及其受体在造血干细胞的体外表达检测

目的体外检测神经营养因子及其受体在造血干细胞的表达情况。方法造血干细胞由小鼠股骨的骨髓细胞培养纯化而来,并用免疫组织化学CD34＋鉴定。RT-PCR的方法在mRNA水平检测造血干细胞表达神经营养因子及其受体的情况。结果造血干细胞可以表达多种神经营养因子及其受体,包括NGF、BDNF、NT-3、EGF、PDGF、CNTF、VEGF、TGF-β1、Trk-A、TrkC,其中NGF、BDNF、NT-3、TrkC有强阳性表达;EGF、PDGF、CNTF、TGF-β1、VEGF、Trk-A有表达但相对较NGF、BDNF、NT-3、TrkC弱;GDNFI、GF-1及TrkB无表达。结论造血干细胞能不同程度的表达多种重要的神经营养因子及其相关受体,可能是一种潜在的移植治疗中枢神经系统损伤的良好细胞来源。     

脊髓半横断损伤后腹角神经元神经生长因子、脑源性神经营养因子、神经营养因子-3和神经营养因子-4表达的变化

目的:探讨大鼠脊髓半横断损伤(htSCI)后脑源性神经营养因子(BDNF)、神经生长因子(NGF)、神经营养因子(NT-3、NT-4)在脊髓腹角神经元表达的早期变化。方法:免疫组织化学ABC法分别染4种神经因子并作阳性细胞计数。结果:NGF主要分布于脊髓腹角神经元的胞核,BDNF、NT-4与NT-3主要分布于胞浆。htSCI前后它们在细胞内的分布范围没有变化。BDNF、NGF与NT-3的3 d在损伤尾侧段脊髓双侧腹角阳性神经元数与对照组相比显著减少。BDNF与NGF的14 d的双侧腹角阳性神经元数量均较正常组明显增多,NT-3与NT-4的14 d~21 d的双侧腹角阳性神经元数量均较正常组明显增多,BDNF7~21 d以及NGF14 d的健侧的阳性神经元数量均分别多于相应的伤侧。结论:内源性BDNF、NGF、NT-3、NT-4增加对脊髓损伤修复具有重要作用,BDNF和NGF在健侧表达的增加说明健侧代偿功能的活跃。     

脂肪源性干细胞修复周围神经损伤潜能的研究

目的:体外原代培养脂肪源性干细胞(adipose-derived stem cell,ADSC),并在细胞水平检测各种神经营养因子(neurotrophic factor,NF)的表达情况,探讨ADSC是否可以作为神经损伤修复的种子细胞。方法:体外原代培养和鉴定ADSC,应用RT-PCR方法检测ADSC细胞神经营养因子,包括神经生长因子(nerve growth factor,NGF)、脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)、神经营养素-3(neurotrophin 3,NT-3);免疫组织化学法检测粘附因子(nerve cell adhesion molecule,NCAM)表达情况。结果:ADSC可以向脂肪和骨两方面分化;RT-PCR结果显示ADSC细胞NGF、BDNF、NT-3 mRNA水平明显升高;同时免疫荧光法检测到了高水平的NCAM。结论:ADSC具有干细胞分化潜能,并且能够表达NF和NCAM。     

Neurotrophins are required for nerve growth during development

Although the requirement of neurotrophins for the prevention of cell death in the peripheral nervous system is well established, their physiological involvement in nerve growth is still unclear. To address this question, we generated a mouse that expresses the green fluorescent protein in post-mitotic neurons, allowing the repeated visualization of all motor and sensory axons during development. We imaged the growth of these axons into the limb bud of day 10.5 embryos. Sensory axons, but rarely motor axons, were targeted to ectopically placed beads containing any of the neurotrophins NGF, BDNF, NT-3 or NT-4/5. Conversely, a combination of function-blocking monoclonal antibodies to NGF, BDNF and NT-3 dramatically inhibited elongation of both sensory and motor axons in the limb bud, indicating that the growth of mixed nerves is dependent upon neurotrophins during development.     

Peripheral nerve regeneration and neurotrophic factors

The role of neurotrophic factors in the maintenance and survival of peripheral neuronal cells has been the subject of numerous studies. Administration of exogenous neurotrophic factors after nerve injury has been shown to mimic the effect of target organ-derived trophic factors on neuronal cells. After axotomy and during peripheral nerve regeneration, the neurotrophins NGF, NT-3 and BDNF show a well defined and selective beneficial effect on the survival and phenotypic expression of primary sensory neurons in dorsal root ganglia and of motoneurons in spinal cord. Other neurotrophic factors such as CNTF, GDNF and LIF also exert a variety of actions on neuronal cells, which appear to overlap and complement those of the neurotrophins. In addition, there is an indirect contribution of GGF to nerve regeneration. GGF is produced by neurons and stimulates proliferation of Schwann cells, underlining the close interaction between neuronal and glial cells during peripheral nerve regeneration. Different possibilities have been investigated for the delivery of growth factors to the injured neurons, in search of a suitable system for clinical applications. The studies reviewed in this article show the therapeutic potential of neurotrophic factors for the treatment of peripheral nerve injury and for neuropathies.     

Collagen scaffolds modified with CNTF and bFGF promote facial nerve regeneration in minipigs

Most experiments of peripheral nerve repair after injury have been conducted in the rodent model but the translation of findings from rodent studies to clinical practice is needed partly because the nerve regeneration must occur over much longer distances in humans than in rodents. The reconstruction of long distance nerve injuries still represents a great challenge to surgeons who is engaged in peripheral nerve surgery. Here we used the functional nerve conduit (collagen scaffolds incorporated with neurocytokines CNTF and bFGF) to bridge a 35 mm long facial nerve gap in minipig models. At 6 months after surgery, electrophysiology assessment and histological examination were conducted to evaluate the regeneration of peripheral facial nerves. Based on functional and histological observations, the results indicated that the functional collagen scaffolds promoted nerve reconstruction. The number and arrangement of regenerated nerve fibers, myelination, and nerve function reconstruction was better in the CNTF + bFGF conduit group than the single factor CNTF or bFGF conduit group. The functional composite conduit, which exhibited favorable mechanical properties, may promote facial nerve regeneration in minipigs effectively.     

LIF、CNTF和BDNF对动眼神经切断后猫动眼神经核CGRP表达的影响

为观察外源性白血病抑制因子(LIF)、睫状神经营养因子(CNTF)和脑源性神经营养因子(BDNF)对动眼神经(ONe)切断后猫动眼神经核(ONu)降钙素基因相关肽(CGRP)表达的影响,本研究采用猫ONe切断后硅胶管套接模型,向再生室内分别给予LIF、CNTF、BDNF、BDNF+LIF、BDNF+CNTF和生理盐水(NS),存活12周后用免疫组织化学方法检测各组动物ONu内CGRP的表达变化。结果发现:各营养因子组动物术侧ONu内的CGRP表达均明显高于NS组(P<0.01)。BDNF+LIF组与BD-NF+CNTF组之间无明显差别(P>0.05),但均明显高于因子单用组(P<0.01);因子单用组中CNTF组与BDNF组之间无明显差别(P>0.05),但均高于LIF组(P<0.05)。以上结果提示,外源性LIF、CNTF和BDNF均可上调ONe切断后猫ONu的CGRP表达,LIF作用较弱,联合应用具有协同作用。     

部分背根切断后备用DRG卫星细胞NGF、BDNF和NT-3的表达变化

为探讨部分去背根猫备用背根节 ( L6 )卫星细胞 NGF、BDNF和 NT-3的表达变化 ,将成年雄猫 2 0只分为正常对照组和术后 3 d、术后 7d及术后 10 d三个实验组。实验组行单侧部分背根切断术 (切除一侧 L1 ～ L5,L7～S2 DRG,保留 L6 背根为备用根 )。取正常组一侧和术后 3 d、7d及 10 d实验组手术侧的 L6 DRG制作 2 0μm厚冰冻切片 ,行免疫组化反应 ;观察 NGF、BDNF和 NT-3在 DRG卫星细胞的分布 ,计数各组 L6 DRG的 NGF、BDNF和 NT-3的阳性卫星细胞数量。结果发现 ,部分去背根术后7d和 10 d组 L6 DRG的 NGF、BDNF和 NT-3的阳性卫星细胞数量较术后 3 d组和正常组者明显增高 ( P<0 .0 1)。表明 ,单侧部分背根切断导致备用 DRG卫星细胞的 NGF、BDNF和 NT-3的表达上调 ,这一变化可能与脊髓可塑性有关。     

Gelatin-tricalcium phosphate membranes immobilized with NGF, BDNF, or IGF-1 for peripheral nerve repair: an in vitro and in vivo study

In the present study, NGF, BNDF from the neurotrophin family and IGF-1 were covalently immobilized on gelatin-tricalcium phosphate (GTG) membrane using carbodiimide. We investigated the effects of these growth factors released from the GTG composites on cultured PC12 cells and sciatic nerve regeneration across a 10-mm-long gap in rats. In PC12 cell culture, the total protein content and MTT assay indicated more cell attachment on the composites modified with growth factors. The IGF-1 group showed a higher survival promotion effect on PC12 cells than did BDNF and NGF groups. On the other hand, NGF released from the composite showed the highest level of neuritogenesis for PC12 cells in neurite outgrowth assay. In the animal study, the GTG conduits modified with various growth factors were well tolerated by the host tissue. In the regenerated nerves, the number of the axons per unit area of the BDNF group was significantly higher than that of NGF and GTG groups but similar to that of IGF-1 group. However, the average axon size was the largest in NGF group. This result was in concordance with the neurite outgrowth assay in which NGF showed the highest neuritogenic potential. In the assessment of motor and sensory recovery after nerve repair, conduits modified with various neurotrophic factors showed a more favorable outcome in compound muscle action potential. The BDNF group had a better gastrocnemic muscle weight ratio than blank GTG repair. Nevertheless, the different effects of GTG conduits modified with various neurotrophic factors on functional recovery cannot be simply illustrated in the sciatic function index.     

An in vivo characterization of trophic factor production following neural precursor cell or bone marrow stromal cell transplantation for spinal cord injury

Cellular transplantation strategies for repairing the injured spinal cord have shown consistent benefit in preclinical models, and human clinical trials have begun. Interactions between transplanted cells and host tissue remain poorly understood. Trophic factor secretion is postulated a primary or supplementary mechanism of action for many transplanted cells, however, there is little direct evidence to support trophin production by transplanted cells in situ. In the present study, trophic factor expression was characterized in uninjured, injured-untreated, injured-treated with transplanted cells, and corresponding control tissue from the adult rat spinal cord. Candidate trophic factors were identified in a literature search, and primers were designed for these genes. We examined in vivo trophin expression in 3 paradigms involving transplantation of either brain or spinal cord-derived neural precursor cells (NPCs) or bone marrow stromal cells (BMSCs). Injury without further treatment led to a significant elevation of nerve growth factor (NGF), leukemia inhibitory factor (LIF), insulin-like growth factor-1 (IGF-1), and transforming growth factor-β1 (TGF-β1), and lower expression of vascular endothelial growth factor isoform A (VEGF-A) and platelet-derived growth factor-A (PDGF-A). Transplantation of NPCs led to modest changes in trophin expression, and the co-administration of intrathecal trophins resulted in significant elevation of the neurotrophins, glial-derived neurotrophic factor (GDNF), LIF, and basic fibroblast growth factor (bFGF). BMSCs transplantation upregulated NGF, LIF, and IGF-1. NPCs isolated after transplantation into the injured spinal cord expressed the neurotrophins, ciliary neurotrophic factor (CNTF), epidermal growth factor (EGF), and bFGF at higher levels than host cord. These data show that trophin expression in the spinal cord is influenced by injury and cell transplantation, particularly when combined with intrathecal trophin infusion. Trophins may contribute to the benefits associated with cell-based repair strategies for spinal cord injury.     

Dexamethasone and vitamin B12 synergistically promote peripheral nerve regeneration in rats by upregulating the expression of brain-derived neurotrophic factor

Introduction

Dexamethasone and vitamin B₁₂ are currently used in the clinic to treat peripheral nerve damage but their mechanisms of action remain incompletely understood. In this study we hypothesized that dexamethasone and vitamin B₁₂ promote the production of endogenous neurotrophic factors, thereby enhancing peripheral nerve repair.

Material and methods

Ninety-six adult male Wistar rats were employed to establish a sciatic nerve injury model. They were then randomly divided into 4 groups to be subjected to different treatment: saline (group A), dexamethasone (group B), vitamin B₁₂ (group C), and dexamethasone combined with vitamin B₁₂ (group D). The walking behavior of rats was evaluated by footprint analysis, and the nerve regeneration was assessed by electrophysiological analysis and ultrastructural examination. The expression of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor, NT-3 and IL-6 in the injured sciatic nerves was detected by immunohistochemical and RT-PCR analysis.

Results

Dexamethasone and vitamin B₁₂ promoted the regeneration of myelinated nerve fibers and the proliferation of Schwann cells. Furthermore, dexamethasone and vitamin B₁₂ promoted the recovery of sciatic functional index and sensory nerve conduction velocity, and upregulated BDNF expression in the injured sciatic nerves.

Conclusions

Dexamethasone and vitamin B₁₂ promote peripheral nerve repair in a rat model of sciatic nerve injury through the upregulation of BDNF expression. These findings provide new insight into the neurotrophic effects of dexamethasone and vitamin B₁₂ and support the application of these agents in clinical treatment of peripheral nerve injury.     

Fibroblast-like cells from rat plantar skin and neurotrophin-transfected 3T3 fibroblasts influence neurite growth from rat sensory neurons in vitro

Our previous finding that skin-derived and muscle-derived molecules can be used to sort regenerating rat sciatic nerve axons evoked questions concerning neuron-target interactions at the level of single cells, which prompted the present study. The results show that dorsal root ganglion (DRG) neurons co-cultured with fibroblast-like skin-derived cells emit many neurites. These have a proximal linear segment and a distal network of beaded branches in direct relation to skin-derived cells. Electron microscopic examination of such co-cultures showed bundles of neurites at some distance from the target cells and single profiles closely apposed to subjacent cells. RNase protection assay revealed that cultivated skin-derived cells express nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). In co-cultures of DRG neurons and 3T3 fibroblasts overexpressing either of the neurotrophins produced by skin-derived cells the picture varied. NT-3 transfected 3T3 fibroblasts gave a growth pattern similar to that seen with skin-derived cells. Neurons co-cultured with mock-transfected 3T3 fibroblasts were small and showed weak neurite growth. In co-cultures with a membrane insert between skin-derived cells or 3T3 fibroblasts and DRG neurons few neurons survived and neurite growth was very sparse. We conclude that skin-derived cells stimulate neurite growth from sensory neurons in vitro, that these cells produce NGF, BDNF, NT-3 and NT-4 and that 3T3 fibroblasts producing NT-3 mimic the effect of skin-derived cells on sensory neurons in co-culture. Finally the results suggest that cell surface molecules are important for neuritogenesis.     

睫状神经营养因子调节受损视神经胶质细胞去分化相关基因的表达

目的 探讨睫状神经营养因子(CNTF)对受损视神经胶质细胞去分化的作用及机制.方法 将65只SD大鼠随机分为对照组、损伤组和CNTF组,制备视神经损伤及损伤后添加CNTF的动物模型.术后7、14 d取术侧视神经损伤远侧段,分别用基因芯片检测其基因表达谱,实时荧光定量PCR、免疫组织化学、HE染色等方法检测相关基因、蛋白表达和细胞数量的变化.结果 术后7 d,与损伤组相比,CNTF组筛选出608条表达上调和417条表达下调的差异基因,包括染色质构型、转录调节、神经干细胞、神经分化和发育、增殖凋亡、离子通道、受体、信号转导等相关基因.实时荧光定量PCR结果验证了基因芯片结果的可靠性.CNTF组视神经损伤远侧段细胞数量增多,远侧段Nestin、胶质纤维酸性蛋白(GFAP)、髓鞘碱性蛋白(MBP)、细胞外信号调节激酶1/2(Erk1/2)和近侧段神经丝(NF)阳性物质增多.结论外源性CNTF通过调节受损视神经大胶质细胞的去分化相关基因及蛋白的表达,促进了胶质细胞的去分化和轴突再生.     

Neurotrophin and Neurotrophin Receptors in Vascular Smooth Muscle Cells: Regulation of Expression in Response to Injury

The neurotrophins, a family of related polypeptide growth factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-3 and NT-4/5 promote the survival and differentiation of distinctive sets of embryonic neurons. Here we define a new functional role for neurotrophins, as autocrine or local paracrine mediators of vascular smooth muscle cell migration. We have identified neurotrophins, and their cognate receptors, the trk tyrosine kinases, in human and rat vascular smooth muscle cells in vivo. In vitro, cultured human smooth muscle cells express BDNF; NT-3; and trk A, B, and C Similarly, rat smooth muscle cells expressed all three trk receptors as well as all four neurotrophins. Moreover, NGF induces cultured human smooth muscle cell migration at subnanomolar concentrations. In the rat aortic balloon deendothelialization model of vascular injury, the expression of NGF, BDNF, and their receptors trk A and trk B increased dramatically in the area of injury within 3 days and persisted during the formation of the neointima. In human coronary atherosclerotic lesions, BDNF, NT-3, and NT-4/5, and the trk B and trk C receptors could be demonstrated in smooth muscle cells. These findings suggest that neurotrophins play an important role in regulating the response of vascular smooth muscle cells to injury.     

Modulation of neurotrophin and neurotrophin receptor expression in nasal mucosa after nasal allergen provocation in allergic rhinitis

Background: Patients with allergic rhinitis (AR) feature both allergic airway inflammation and a hyperresponsiveness to nonspecific stimuli which is partly neuronally controlled. Still, it is unclear whether or not neurotrophins are involved in airway pathophysiology of AR and in nasobronchial interaction. Methods: Nine AR patients with mono‐allergy to grass pollen and nine healthy controls underwent nasal allergen provocation (NP). Serum samples, nasal and bronchial biopsies were taken before (T₀) and 24 h after (T₂₄) NP. Pan‐neurotrophin receptor p75^NTR, tyrosine kinase A (trkA), trkB, nerve growth factor (NGF), and brain‐derived neurotrophic factor (BDNF) were assessed with immunohistochemistry, and NGF and BDNF levels with ELISA. Results: At T₂₄, BDNF and NGF were upregulated in nasal mucosa (P < 0.05) and increased in the peripheral blood of AR compared with T₀. The increase in nasal BDNF expression correlated positively with the maximum increase in total nasal symptom score in AR (P = 0.02). p75^NTR was expressed on peripheral nerves and epithelial layer, trkA on endothelial cells, and trkB on mast cells. trkB + mast cells significantly decreased after NP in AR (P < 0.01). NP did not modulate p75^NTR and trkA expression in nasal mucosa and had no effect on the expression of neurotrophins and receptors in bronchial mucosa. Conclusion: This study shows that neurotrophins and their receptors are expressed in human airways. Allergic rhinitis was characterized by a modulation of BDNF, NGF, and trkB in nasal mucosa after NP and a correlation of nasal BDNF with the maximal increase of total nasal symptom score. Therefore, our data suggest that neurotrophins participate in upper‐airway pathophysiology in AR, whereas their role in nasobronchial interaction remains unclear.     

Intracranial transplantation of human adipose-derived stem cells promotes the expression of neurotrophic factors and nerve repair in rats of cerebral ischemia-reperfusion injury

Adipose tissue-derived mesenchymal stem cells (ADSCs) are of great interest as a cellular therapeutic agent for regenerative and immunomodulatory purposes. The aim of this study was to investigate whether ADSCs transplantation could promote nerve repair in rats of cerebral ischemia-reperfusion (I/R) injury. We isolated and cultured human ADSCs, and then measured cell surface antigens by flow cytometry and immunofluorescence. Healthy SD rats were randomly divided into sham group, MCAO group, MCAO+vehicle group and MCAO+ADSCs group. Cerebral ischemia-reperfusion injury was induced by middle cerebral artery occlusion (MCAO). Then the human ADSCs were transplanted into the brain of rats 24 h after MCAO. The mRNA level of BDNF (brain derived neurotrophic factor, BDNF), NGF (nerve growth factor, NGF) and bFGF (basic fibroblasts growth factor, bFGF) were detected by real-time PCR at different time points (d7, d14, d21 and d28 after MCAO). Meanwhile, the neurological deficit scores were estimated. The neurological deficit of rats in MCAO+ADSCs group attenuated at d7 in contrast to the MCAO+vehicle group (P<0.05). Subsequently, they were dramatically ameliorated with the time especially at d28. At d7, d14, d21 and d28 after ADSCs transplantation, BDNF, NGF and bFGF mRNA in MCAO+ADSCs group were strikingly higher than those in MCAO+vehicle group, and these two groups both reached the peak at d14. The western blotting results showed that BDNF and Bcl-2 expressed higher in MCAO+ADSCs group than MCAO+vehicle group. Therefore, our current results suggest that ADSCs promote nerve repair after injury through elevating the expression of neurotrophic factors and inhibiting the apoptosis of neural cells.     

Investigating the synergistic effect of combined neurotrophic factor concentration gradients to guide axonal growth

Neurotrophic factors direct axonal growth toward the target tissue by a concentration gradient, which is mediated through different tyrosine kinase cell surface receptors. In this study, well-defined concentration gradients of neurotrophic factors (NFs) allowed us to study the synergistic effect of different NFs (e.g. nerve growth factor [NGF], neurotrophin-3 [NT-3] and brain-derived neurotrophic factor [BDNF]) for axonal guidance of embryonic lumbar dorsal root ganglion cells (DRGs). Effective guidance of DRG axons was achieved with a minimum NGF concentration gradient of 133 ng/ml/mm alone, or combined NGF and NT-3 concentration gradients of 80 ng/ml/mm each. Interestingly, the combined concentration gradients of NGF and BDNF did not show any significant synergism at the concentration gradients studied. The synergism observed between NGF and NT-3 indicates that axons may be guided over a 12.5 mm distance, which is significantly greater than that of 7.5 mm calculated by us for NGF alone or that of 2 mm observed by others.