神经营养素受体p75NTR介导凋亡的机制及其在眼科的研究

p75NTR是一个跨膜糖蛋白，是属于TNF受体超家族成员。NGF、BDNF、NT—3、NT—4／5都可以与p75NTR结合。表达可诱导神经细胞凋亡，可能的途径是激活鞘磷脂通路和转录因子NF-κB。另外还有一些功能相关蛋白参与p75NTR信号传递。p75NTR与某些视网膜神经细胞凋亡关系密切，相关研究已逐渐成为热点。     

p75 NTR受体在视网膜色素上皮细胞氧化损伤中的作用及机制

目的：研究p75 NTR受体在视网膜色素上皮细胞(retinal pigment epithelial cells,RPE)氧化损伤过程中的作用及机制。

方法：将转染p75 NTR受体的RPE细胞作为实验组,未转染的RPE细胞作为对照组。BrdU检测法检测细胞增殖活性; PI/Annexin V-FITC双染法检测细胞凋亡率; 激光显微镜观察细胞内ROS的表达情况; 流式细胞仪检测细胞内ROS、线粒体标志物、细胞色素C表达水平; Western blot法检测细胞中Fas蛋白、裂解Caspase-3、VEGF165蛋白的表达水平。

结果：随着p75 NTR受体转染时间的延长,实验组RPE细胞的增殖活性呈逐渐降低趋势,各转染时间点的RPE细胞增殖活性比较,差异有统计学意义(P<0.05); 实验组各转染时间点的RPE细胞增殖活性均明显低于对照组,差异均有统计学意义(P<0.05)。随着转染时间的延长,实验组RPE细胞凋亡率呈逐渐增加趋势,各转染时间点的RPE细胞凋亡率比较,差异有统计学意义(P<0.01); 实验组各转染时间点的RPE细胞凋亡率均明显高于对照组,差异均有统计学意义(P<0.01)。实验组ROS荧光信号明显强于对照组。流式细胞仪检测结果显示,实验组RPE细胞中ROS、细胞色素C水平均明显高于对照组,线粒体标志物水平明显低于对照组,差异均有统计学意义(P<0.01)。Western blot 法检测结果表明,实验组细胞内Fas蛋白、Caspase-3、VEGF₁₆₅蛋白的表达水平均明显高于对照组,差异均有统计学意义(P<0.01)。

结论：p75 NTR受体高表达可导致RPE细胞线粒体发生损伤,同时促进细胞凋亡,最终导致脉络膜新生血管的形成,表明p75 NTR受体可能是导致RPE细胞发生损伤的因素之一。     

神经生长因子与高原眼底病

神经生长因子(nerve growth factor,NGF)为神经系统最为重要的神经营养因子之一.在眼科方面,NGF对视网膜神经节细胞的存活及视神经纤维的再生起着极其重要的作用,而高原低氧环境可抑制NGF在视网膜中的表达,影响视网膜修复.本文就NGF及其受体在视网膜的表达、对视网膜的保护作用、高原眼底病以及高原环境下NCF的视网膜表达特征作一综述,并讨论NCF在高原眼底病治疗中的应用前景.     

神经营养素家族因子对视网膜神经细胞保护作用的研究进展

现有大量研究表明神经营养素家族因子对视网膜神经细胞具有重要的保护作用。在所有家族成员中,神经生长因子能够有效保护视网膜光感受器细胞;阻断视网膜神经节细胞中神经生长因子与受体p75的结合,可以抑制神经节细胞的凋亡。脑源性神经营养因子可以防止光感受器细胞变性,增强光感受器细胞损伤后的修复。在刺激神经节细胞轴突生长和突触形成方面,脑源性神经营养因子、神经营养素-3及神经营养素-4/5均具有显著效应。通过对神经营养素家族因子的研究,了解其作用机制,以期能够应用于视网膜神经细胞变性及损伤性疾病的治疗中。     

视网膜光化学损伤感光细胞凋亡的分子基础

视网膜光化学损伤动物模型是研究视网膜变性类疾病的良好模型，研究发现凋亡是视网膜感光细胞光化学损伤以及其它视网膜变性疾病感光细胞丢失的主要机制。本文阐述了核转录因子κB（NFκB）体系，arrestin蛋白家族，AP-1和神经营养因子受体P75NTR等调控感光细胞凋亡的分子机制。 （中华眼底病杂志，2004，20：396-398）     

神经生长因子对兔准分子激光角膜磨镶术后角膜神经再生的作用研究

干眼症是准分子激光角膜磨镶术（laser in situ keratomileusis，LASIK）术后最常见的并发症，其发生原因诸多，主要与术中做角膜瓣时切断了除角膜瓣蒂部以外的所有神经，从而导致角膜感觉降低，影响泪液分泌。因此，促进角膜知觉的恢复，即促进LASIK术后角膜神经纤维的再生与修复是治疗LASIK术后干眼的有效方法。神经生长因子（nerve growth factor，NGF）是神经系统最重要的生物活性分子之一，是参与损伤神经再生和功能修复的重要因素。NGF是通过受体介导而发挥作用，在角膜上皮细胞、内皮细胞及基质细胞都存在NGF受体。本研究旨在探讨外源性NGF对角膜神经损伤再生与修复作用。     

神经生长因子对吲哚美辛诱导人胚胎视网膜色素上皮细胞凋亡的保护作用

目的 探讨神经生长因子(nerve growth factor, NGF)对体外培养的人胚胎视网膜色素上皮(human fetal retinal pigment epithelium, HFRPE)细胞凋亡的保护作用。 方法 用传代培养的HFRPE细胞，建立吲哚美辛(indome thacin, IN)诱导的细胞凋亡模型，并用吖啶橙(acridine orange, AO)荧光染色计数和透射电镜(transmission electron microsocpy,TEM)观察NGF对HFRPE细胞凋亡的保护作用。 结果 用AO荧光染色及TEM均观察到经200 ～600 μmol/L IN处理24 h后的HFRPE细胞出现典型的凋亡形态学改变。200 μmol/L IN+500 μg/L NGF组与对照组200 μg/(mol·L) IN相比凋亡细胞差异有显著性的意义(q=3.9204 ，P=0.0320)；400 μmol/L IN+500 μg/L NGF组与对照组400 μmol/L IN相比凋亡细胞差异有非常显著性的意义(q=9.709 15，P=0.000 1)。 结论 NGF 能拮抗IN诱导的HFRPE细胞凋亡。 （中华眼底病杂志，2003，19：38-41）     

P75神经营养受体与视网膜细胞凋亡

P75神经营养受体是最早发现的神经营养因子受体，既往认为其主要作为高亲和力受体的辅助受体间接发挥作用。近年来研究表明，P75神经营养受体在神经系统的发育中有诱导细胞凋亡作用。现阐述其在视网膜神经细胞生长，分化以及逆向运输神经营养因子等方面的作用。     

实验性视网膜脱离时神经生长因子对视网膜细胞凋亡的干预作用

目的 研究神经生长因子（NGF）在实验性视网膜脱离（RD）时对视网膜细胞凋亡的干预作用。 方法 27只大鼠左、右眼分别被分为实验对照组和NGF组。视网膜下注射透明质酸钠建立RD动模型后，取右眼在玻璃体腔内注射1μg/μl NGF，共5μl，作为NGF组；左眼注射磷酸盐缓冲液（PBS）5μl，作为实验对照组。注射方法为每4天注射1次，直至观察期结束。两组在建立模型后1.5、3、6、12h、1 、2、4、8、16、32d分别取眼球。另设立正常对照组2只大鼠，不作任何处理，在实验观察结束时取眼球。采用原位末端标记法(TUNEL)和透射电子显微镜观察视网膜细胞凋亡情况,并进行细胞计数和统计学检验。 结果 RD早期就有细胞凋亡的发生。视网膜各层细胞均可见凋亡细胞，内、外核层分布最多。凋亡细胞数随脱离时间延长而增加（P<0.01）。NGF组的凋亡细胞数比实验对照组少（P<0.01）。 结论 实验性RD中，玻璃体腔内给予外源性NGF能抑制视网膜细胞凋亡的发生。 (中华眼底病杂志, 2006, 22: 333-335)     

神经生长因子对人胚胎视网膜色素上皮细胞生长及其DNA合成的影响

研究表明一些细胞因子(如碱性成纤维细胞生长因子等)在体外能促进培养的视网膜色素上皮细胞 (retinal pigment epithelium, RPE) 增生和DNA合成[1].神经生长因子(nerve growth factor, NGF)不同于其他细胞因子,是主要作用于神经细胞和神经系统生长、发育以及发挥正常生理功能所必须的一类神经营养因子,虽然 RPE 细胞不是神经细胞,但它是从神经外胚层分化而来的,因此,我们试图在体外探讨NGF是否对 RPE 细胞生长以及DNA合成有促进作用.     

Nerve growth factor effect on human primary fibroblastic-keratocytes: possible mechanism during corneal healing

In response to corneal injury, cytokines and growth factors play a crucial role by influencing epithelial-stromal interaction during the healing and reparative processes which may resolve in tissue remodeling and fibrosis. While transforming growth factor-beta1 (TGF-beta1) is considered the main profibrogenic modulator of these process, recently the nerve growth factor (NGF) appears as a pleiotropic modulator of wound-healing and inflammatory responses. Interestingly in the cornea, where NGF, trkA(NGFR) and p75(NTR) are expressed by epithelial cells and keratocytes, the NGF eye-drop induces the healing of neurotrophic or autoimmune corneal ulcers. During corneal healing, quiescent keratocytes are replaced by active fibroblast-like keratocytes/myofibroblasts. While the NGF effect on epithelial cells has been investigated, no data are reported for NGF effects on fibroblastic-keratocytes, during corneal healing. NGF, trkA(NGFR) and p75(NTR) were found expressed by fibroblastic-keratocytes. NGF was able to induce fibroblastic-keratocyte differentiation into myofibroblasts, migration, Metalloproteinase-9 expression/activity and contraction of a 3D collagen gel, without affecting their proliferation and collagen production. These data also show a two-directional control of fibroblastic-keratocytes by NGF and TGF-beta1. To sum up, the findings of this study indicate that NGF can modulate some functional activities of fibroblastic-keratocytes, thus substantiating the healing effects of NGF on corneal wound-healing.     

Human idiopathic epiretinal membranes express NGF and NGF receptors

PURPOSE: Glial cells and fibroblasts (FBs) play a key role in epiretinal membrane (ERM) development and progression. Myofibroblasts (myoFBs), arising from these cells, can lead to the hypertrophic scars and tissue contraction observed in ERMs. Nerve growth factor (NGF) and transforming growth factor-beta1 (TGF-beta1) play a crucial role in FB activities. Therefore, the authors evaluated myoFBs in ERMs and NGF, trkA(NGFR and p75(NTR) expression, as well as TGF-beta1/TGF-betaRII levels in both ERMs and vitreous. METHODS: Eight idiopathic ERMs and vitreous were obtained from patients at the time of vitrectomy for macular pucker. Ten control vitreous were from donors. Biochemical and molecular analyses were performed to identify alpha-smooth muscle actin (alpha-SMA, a defined myoFB marker), NGF, trkA(NGFR)/p75(NTR), and TGF-beta1/TGF-betaRII. RESULTS: Every idiopathic ERM displayed alpha-SMA positive myoFBs, expressing NGF, trkA(NGFR), and p75(NTR). ERM vitreous showed a significant decrease in NGF protein coupled with a TGF-beta1 increase. In addition, vitreous cells showed an increase in trkA(NGFR)/p75(NTR) mRNA associated with a decrease in TGF-betaRII mRNA. CONCLUSIONS: Idiopathic ERMs were characterized by myoFBs. The expression of NGF, trkA, and p75 in local myoFBs associated with changes in ERM vitreous NGF suggests an involvement of NGF, as previously reported for TGF-beta1, in the evolution and myoFB-mediated contractile activity of ERMs.     

The role of NGF signaling in human limbal epithelium expanded by amniotic membrane culture

PURPOSE: Amniotic membrane (AM) transplantation facilitates rapid epithelialization in severe neurotrophic corneal ulcers. To elucidate its action mechanism, we investigated the expression of ligands and receptors of the neurotrophin family by human limbal epithelial (HLE) cells expanded on AM cultures. METHODS: Expression of nerve growth factor (NGF); neurotrophins (NT)3 and NT4; brain-derived neurotrophic factor (BDNF); tyrosine kinase-transducing receptors TrkA, TrkB, and TrkC; and a pan-NT low-affinity receptor (p75(NTR)) was examined by immunostaining in the normal human corneolimbus, HLE grown on intact epithelially denuded AM, and stratified HLE, after subcutaneous implantation in NIH-bg-nu-xid BR mice. NGF protein level was assayed by an ELISA in extracts of intact and epithelially denuded AM. K252a, a specific inhibitor of TrkA autophosphorylation, was added to test whether it would inhibit HLE expansion on AM culture. RESULTS: Strong positive TrkA staining was confined to the basal epithelial cell layer of normal corneal and limbal epithelia, with the highest intensity noted in the limbus. TrkA staining was also strongly positive in the basal layer of HLE cells cultured on intact and epithelially denuded AM and in basal and some suprabasal layers of stratified HLE transplanted in nude mice. Positive staining of p75(NTR) was noted in the full-thickness of the corneal epithelium but was limited to the superficial layers of the limbus and in HLE cells cultured on intact and epithelially denuded AM, but was weak in HLE transplanted to nude mice. Weak staining of NT3 and TrkC was noted in the suprabasal layers of corneal and limbal epithelia but was negative in the stratified HLE in nude mice. Negative staining of NGF, NT4, BDNF, and TrkB was noted in all specimens tested. The NGF protein level was readily measured as 35.6 +/- 9.1 and 41 +/- 12.5 pg/mg protein in the homogenate of the intact and epithelially denuded AM, respectively (P = 0.0256). K252a significantly inhibited the HLE outgrowth on intact AM culture (P = 0.024). CONCLUSIONS: The strong expression of TrkA but not p75(NTR) in the limbal basal epithelial cells in vivo suggests that NGF signaling favors limbal epithelial stem cell survival. Such a phenotype is preserved in HLE cells on AM. Blocking NGF signaling significantly retarded HLE expansion on AM, supporting the notion that NGF is important in expansion of limbal epithelial progenitor cells. Furthermore, a high and therapeutic level of NGF was present in AM. Collectively, these findings indicate that denervated neurotrophic ulcers are associated with poor epithelial stem cell function at the limbus. Future studies are needed to determine whether AM transplantation to heal such ulcers may include the promotion of nerve regeneration and survival of epithelial progenitor cells.     

Colocalization of TrkB and brain-derived neurotrophic factor proteins in green-red-sensitive cone outer segments

PURPOSE: To examine the distribution of neurotrophins (NTs) and their catalytic receptors in adult rat photoreceptors. METHODS: Immunocytochemistry and Western blot analyses were performed using primary antibodies raised against NTs (nerve growth factor [NGF], brain-derived neurotrophic factor [BDNF], NT-3, and NT-4/5) and NT receptors (TrkA, TrkB, TrkC, and p75NTR). Double-labeling of retinal sections with opsin-specific antibodies was performed to identify each photoreceptor type. Competitive experiments using excess recombinant NT or Trk receptors confirmed the binding specificity of each antibody. RESULTS: TrkB and BDNF immunoreactivity was colocalized in cone outer segments. TrkB and BDNF were detected in all green-red-sensitive cones, but not in blue-UV cones or rods, and other NTs and NT receptors were not detected in any of the photoreceptor types. CONCLUSIONS: The findings suggest a specific role for BDNF through its signaling receptor TrkB in the function and maintenance of green-red cones, the predominant cone type in the rat retina.     

神经生长因子细胞保护作用的应用研究进展

神经生长因子（ＮＧＦ）因具有神经营养和促进轴突生长的双重效应，近年来已较多应用于神经科和眼科的基础和临床治疗研究。外节变性和视细胞凋亡是视网膜脱离术后视力不良的一个重要原因，而视网膜是神经系统的一部分，ＮＧＦ或许可用于视网膜脱离后视细胞凋亡的治疗。综述了ＮＧＦ在神经系统和视网膜中的细胞保护作用、机制及应用方面的进展，为其进一步用于视网膜脱离后抗视细胞凋亡治疗提供理论基础。     

Function of glial cell network as a modulator of neural cell death during retinal degeneration

PURPOSE: Recent studies have demonstrated the mechanism of neural cell death, neuroprotection, and regeneration. However, the functional importance of glial cells during retinal degeneration is not well understood. In this review, we summarize our recent progress regarding the function of glial cells in neurotrophic factor production and neural cell death during retinal degeneration. METHODS: We made a rat model of photoreceptor degeneration by continuous light exposure, and examined the distribution and expression levels of neurotrophins and their receptors. In addition, we carried out quantitative analysis of neurotrophic factor production in cultured Müller glial cells and microglia. RESULTS: In the light-degenerated retina, microglia invade the photoreceptor layer from the inner part of the retina and increase the production of nerve growth factor (NGF). NGF decreases the production of basic fibroblast growth, factor, which prevents photoreceptor cell death, in Müller glial cells through low-affinity neurotrophin receptor p 75. Blockade of p 75 decreased photoreceptor cell death during light-induced retinal degeneration. CONCLUSIONS: These results suggest that a gliaglia network plays a critical role in neural cell death during retinal degeneration. Thus, a glia-glia network as well as a glia-neuron network could be a possible therapeutic target for inhibition of retinal degeneration.