视网膜中神经生长因子及其受体作用研究新进展

神经生长因子对神经系统发育过程中的生长、生存及成熟神经系统的维持都是必不可少的。但也有相反的报道 ,NGF可通过神经营养素低亲和型受体 p75 NTR诱导细胞凋亡的发生。我们综述了NGF这两方面的内容 ,着重介绍神经生长因子及其受体在视网膜的分布和作用以及p75 NTR介导细胞凋亡的机制。     

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。     

神经营养因子和生长因子等对培养的成人视网膜神经细胞的影响

目的　探讨成人视网膜神经细胞体外培养条件 ,脑源性神经营养因子 (BDNF)、神经营养素 (NT 4 )、表皮生长因子 (EGF)、成纤维细胞生长因子 (FGF)、诱导分化因子全反式视黄酸 (RA)等对成人视网膜神经细胞生长、增殖、凋亡的影响及调控机制。方法　胰蛋白酶消化结合机械吹打分离成人视网膜神经细胞 ,在培养基中加入或不加入BDNF、NT 4、EGF、FGF、RA。根据细胞形态、生长方式及免疫细胞化学特征确定细胞类型。比较各组中神经元的数目、转录调控因子c fos、c jun及细胞凋亡调控因子Bcl 2、Bax表达水平。结果　与对照组相比 ,BDNF、FGF处理组存活的神经元特异性烯醇酶、Thy1.1抗体和Bcl 2、c fos及c jun表达阳性细胞数也增多 (均P <0 0 1) ,培养的视网膜神经细胞在体外存活时间可长达 8个月 ;RA处理组c fos、c jun阳性细胞数较对照组增多 (均P <0 0 1) ;而NT 4、EGF处理组各项指标与对照组比较 ,差异无显著意义 (均P >0 0 5 )。结论　BDNF、FGF、RA能显著提高体外培养的成人视网膜神经细胞的存活 ,其机制可能涉及上调转录调控因子c fos、c jun及凋亡抑制因子Bcl 2的表达 ,或下调凋亡促进因子Bax的表达。但EGF、NT 4对体外培养的视网膜神经细胞存活状态无明显改善作用。     

影响视网膜神经节细胞再生的信号分子

已经证实视网膜神经节细胞(RGCs)在一定条件下可以再生，近年来很多学对RGCs的再生做了大量研究，发现Bcl-2、微管结合蛋白、热休克蛋白90、生长相关蛋白43、神经微丝、低亲和性神经营养因子受体p75NTR、cAMP、E587Ag信号分子的表达与RGCs的再生关系密切。本就促进RGCs再生的信号分子作一综述，为进一步的研究提供参考。     

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）     

影响视网膜神经节细胞再生的信号分子

已经证实视网膜神经节细胞(RGCs)在一定条件下可以再生,近年来很多学者对RGCs的再生做了大量研究,发现Bcl-2、微管结合蛋白、热休克蛋白90、生长相关蛋白43、神经微丝、低亲和性神经营养因子受体p75NTR、cAMP、E587Ag信号分子的表达与RGCs的再生关系密切。本文就促进RGCs再生的信号分子作一综述,为进一步的研究提供参考。     

眼眶横纹肌肉瘤细胞凋亡及凋亡相关基因Bcl-2,Bax,p53的蛋白表达

目的 分析眼眶横纹肌肉瘤中的细胞凋亡及凋亡相关基因Bcl-2，Bax和p53的表达状态。方法 对31例标本，利用TUNEL技术显示凋亡细胞，用免疫组织化学方法检测Bcl-2，Bax及p53蛋白表达。结果 凋亡细胞检测率90．3％，肿瘤增生活跃区多见。Bcl－2阳性率29％，Bax阳性率54．5％，p53阳性率64．5％。Bcl-2与Bax表达呈正相关（P＜0．01）。Bcl-2阳性细胞与凋亡细胞分布区域明显不同。Bax与p53表达与细胞凋亡无相关关系（P＞0．05）。结论 眼眶横纹瘤中存在细胞凋亡与肿瘤增生有关。Bcl－2抑制Bax的功能进而抑制肿瘤细胞凋亡。p53在该肿瘤中可能不是调控细胞凋亡的主要因素。     

小鼠视网膜缺血-再灌注后核因子-κB的激活

目的 研究小鼠视网膜缺血-再灌注所致视网膜神经细胞凋亡中,核因子-κB的表达。方法 通过升高小鼠眼内压造成视网膜缺血,用计算机图像分析方法测量视网膜再灌注后神经细胞凋亡的比例和视网膜厚度的改变。免疫组化标记核因子-κB p65亚单位,并与原位缺口末端标记（TUNEL）做双重荧光标记,分析核因子-κB的表达与细胞凋亡之间的时相关系。结果 视网膜缺血-再灌注后最初24h,视网膜内层厚度增加,至168h,厚度显著减少。再灌注后6h,神经节细胞和内核细胞层中p65的免疫表达增强,至24h达到高峰,这一过程与TUNEL标记的时相一致。结论 视网膜缺血-再灌注损伤后,核因子-κB的激活对于视网膜神经细胞的凋亡有重要作用,对于其起促进凋亡还是抑制凋亡的作用,则有待于进一步研究。     

视网膜神经细胞生长发育中相关诱导因素的研究进展

视网膜神经细胞在生长发育过程中受多种因素影响，涉及神经细胞间、神经细胞与基质间、神经细胞和细胞外因子之间作用的复杂过程。脑源性神经营养因子和胶质细胞源性神经营养因子等神经营养因子在神经细胞发育中能够促进细胞存活和增殖，保护神经细胞功能；成纤维细胞生长因子和血小板源性生长因子等细胞因子对细胞生长有直接或间接作用，介导和调节细胞的生物学效应，改变微环境平衡，对视网膜神经细胞生长、分化和凋亡有着促进或抑制作用。[眼科新进展2007；27（2）：146—149]     

糖尿病视网膜病变与细胞凋亡关系的研究进展

糖尿病视网膜病变与细胞凋亡的研究近几年有许多相关文献发表，研究结果表明，糖尿病视网膜病变的发生发展与细胞凋亡有着密切的联系。在细胞凋亡方面的研究，大体可以分为内皮细胞、周细胞、神经细胞三大研究方面。细胞凋亡的研究为从根本上了解糖尿病视网膜病变的发病机制提供了新的思路及方法，为治疗糖尿病视网膜病变开辟了新的途径。     

Lack of p75 receptor does not protect photoreceptors from light-induced cell death

Rod photoreceptors are susceptible to light-induced cell death. Previous results have suggested that the neurotrophin receptor p75 in Müller cells controls photoreceptor cell death during light-exposure by suppressing trophic factor release; and consequently, if p75 is blocked or eliminated during light-exposure, apoptosis is delayed. We explored this question by examining photoreceptor cell survival in albino p75(-/-) mice as well as their heterozygous and homozygous littermates. Photoreceptor cell death was examined in semi-thin sections by counting the remaining rows of photoreceptors. No difference in the amount of cell death was found between p75(+/+) and p75(-/-) animals, whereas the single copy of p75 in the heterozygous p75(+/-) mice provided significant neuroprotection. Cell death in the wild-type animals may indeed be mediated by p75, whereas other known apoptosis pathways may be activated in the p75(-/-) mice. The pro-apoptotic activity of the p75 receptor may have been partially suppressed in the heterozygous p75(+/-) mice by the silencing effect of the Trk receptor. Thus, our results suggest that p75 signaling does not mediate the main apoptosis pathway activated during light-damage.     

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.     

Role of neurotrophin-4/5 in neural cell death during retinal development and ischemic retinal injury in vivo

PURPOSE: Neurotrophin (NT)-4/5 and brain-derived neurotrophic factor (BDNF) mediate cell survival through TrkB, a high-affinity tyrosine kinase receptor, and may prevent neural cell death in various pathologic conditions. This study was conducted to investigate the function of NT-4/5 in neural cell death during retinal development and ischemic retinal injury. METHODS: Retinal development in wild-type, NT-4/5 knockout (KO), and NT-4/5:BDNF double-KO mice was histologically examined from postnatal day 0 (P0) to P90. Ischemic retinal injury was performed at P42, and NT-4/5 mRNA expression level and the extent of retinal cell death was quantitatively examined. RESULTS: Real-time PCR analysis revealed increased NT-4/5 mRNA expression in the ischemic retina. In the NT-4/5 KO mouse, retinal development and structure were normal, but the strain was susceptible to ischemic injury on P42. In contrast, NT-4/5:BDNF double-KO mice showed delayed retinal development and died before P42. CONCLUSIONS: These results suggest that NT-4/5, in combination with other trophic factors, is involved in the postnatal survival of retinal neurons during both development and degeneration.     

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.     

In vivo expression of neurotrophins and neurotrophin receptors is conserved in adult porcine retina in vitro

PURPOSE. To characterize and compare the expression of neurotrophins (NTs) and their receptors within adult porcine retinal ganglion cells (RGCs) in vivo and in vitro. METHODS. The distribution of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and -4 (NT-4), and their high-affinity receptors TrkA, TrkB, TrkC and low-affinity receptor p75, was analyzed in adult porcine retinal sections by immunohistochemistry. In addition, adult porcine retinas were dissociated and cultured in four different conditions: control, semipure RGCs, supplemented with BDNF, or seeded on Müller glia feeder layers. Double immunostaining was performed with antibodies to NTs or their receptors combined with neurofilament antibody to identify RGCs in culture. RESULTS. In vivo, immunolabeling of NGF was very faint, BDNF was especially prominent in RGCs and inner nuclear layer cells, NT-3 stained widespread nuclei, and NT-4 was undetectable. TrkA immunoreactivity was visible in the nerve fiber layer, TrkB staining was within RGC bodies, TrkC was undetectable, and p75 was widely expressed across the retina, within the Müller glia. Expression of neurotrophins and their receptors was maintained in all four models of adult RGCs in vitro, showing that expression was not influenced by substrate or culture conditions. We observed prominent staining of TrkA within growth cones, and a clear expression of p75 within a subpopulation of RGCs in vitro. CONCLUSIONS. These findings demonstrate that the expression of NTs and their receptors within adult porcine RGCs is maintained in vitro, under conditions of limited interaction with neighboring neurons and deprived of afferent inputs and target tissue. TrkA may be involved in regeneration of nerve terminals.     

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.     

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.