NGF及其受体trkA、p75在翼状胬肉中的实验研究
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摘要
第一部分NGF及其受体trkA、p75在翼状胬肉组织中的表达
目的研究翼状胬肉组织中的NGF及其受体trkA、p75蛋白表达，探讨其与翼状胬肉形成的关系。
方法应用免疫组织化学检测30例翼状胬肉组织以及5例正常结膜组织中NGF、trkA和p75蛋白的表达。
结果NGF、trkA和p75蛋白在翼状胬肉组织的上皮细胞、成纤维细胞、血管内皮细胞中呈阳性表达；NGF、trkA蛋白在正常结膜组织上皮细胞和成纤维细胞中呈弱阳性表达；p75蛋白仅在正常结膜组织上皮细胞中呈弱阳性表达。
结论NGF、trkA、p75可能共同参与翼状胬肉的发生、发展过程。
第二部分p75和α-SMA在翼状胬肉组织的免疫定位
目的检测p75和α-SMA在翼状胬肉组织的表达定位。
方法正常结膜组织3例、翼状胬肉组织10例来自手术切除，3例正常角膜组织来自眼库。福尔马林浸泡并石蜡包被切片，用免疫组化方法检测trkA、p75和α-SMA在各种组织中的表达。利用共聚焦荧光显微镜通过免疫双标的方法检测p75和α-SMA在翼状胬肉组织及原代成纤维细胞中的定位。
结果p75、trkA和α-SMA在翼状胬肉组织成纤维细胞中表达明显。在翼状胬肉组织、正常角膜和结膜组织中检测到p75和trkA的表达。p75和α-SMA在翼状胬肉组织的头部可见阳性表达，在体部阳性表达较少。
结论在翼状胬肉组织头部p75和α-SMA表达在同一部位，p75表达上调可能参与细胞的增殖。
第三部分β-NGF对人翼状胬肉成纤维细胞增殖的影响
目的观察β-NGF(Nerve growth factor)对体外培养人翼状胬肉成纤维细胞(human pterygium fibroblasts，HPF)增殖的影响，探讨翼状胬肉发病机制。
方法免疫荧光法检测HPF中β-NGF受体trkA、p75的表达，MTT法检测不同浓度β-NGF对HPF的作用，Western-blot及RT-PCR法检测细胞核抗原(PCNA)来评估HPF增殖。结果trkA、p75在HPF中呈阳性表达；48h为β-NGF促增殖的高峰期；β-NGF在促HPF增殖的影响呈剂量依赖性。
结论β-NGF可能通过结合两受体trkA、p75促进了HPF的增殖。
第四部分trkA抑制剂对人翼状胬肉成纤维细胞凋亡的影响
目的探讨trkA抑制剂对体外培养人翼状胬肉成纤维细胞(HPF)生长抑制及其机制。
方法免疫荧光法检测HPF中β-NGF受体trkA、p75的表达，10～80nmol／LtrkA抑制剂作用24～96h后，MTF比色法检测细胞生长活性，分光光度法检测caspase-3在细胞中的表达，流式细胞仪测定细胞凋亡。
结果trkA抑制剂能显著抑制HPF的体外生长，呈剂量和时间依赖性；caspase-3活性检测表达增高，凋亡率为8．26％～29．62％(P＜0．01)。
结论trkA抑制剂可能通过改变HPF中trkA和p75的比率上调caspase-3诱导细胞凋亡。
Objective To investigate the expression of NGF, trkA and p75 in pterygium, and toanalyse their correlation with form of pterygium.
Methods NGF, trkA and p75 expression was detected in 30 pterygium tissues and 5normal conjunctiva tissues by immunohistochemical method.
Results Expression of NGF, trkA and p75 was significantly observed in epithelium,fibroblasts and vascular endothelial cells of pterygium tissues. Expression of NGF and trkAwas observed in epithelium and fibroblast of normal conjunctiva. Expression of p75 wasonly observed in epithelium of normal conjunctiva.
Conclusion NGF, trkA and p75 possibly participate in genesis and development ofpterygium.
Objective To determine localisation of p75 andα-SMA (α-smooth muscleactin) in pterygium tissues.
Methods Three normal conjunctiva and ten pterygium were surgically collected,and three normal cornea were collected from eye bank. Formalin fixed and paraffinembedded tissue sections were incubated with trkA, p75 polyclonal antibodies andα-SMA monoclonal antibody, and then examined by immunohistochemistry.Immunolocalisation of p75 andα-SMA was detected in pterygium and primarycultured fibroblasts by confocal microscopy.
Results Expression of p75,α-SMA and trkA was detected significantly infibroblasts of pterygium, p75 and trkA was examined in epithelium cells of pterygium,normal conjunctiva and cornea. In the pterygium head, p75 as well asα-SMA washeterogeneously expressed in cytoplasm of fibroblasts, whereas the expression was lessmarked in the body.
Conclusion It is suggested that p75 andα-SMA were concentrated in the sameposition of pterygium head, and p75 was possibly involved in cell proliferation.
Objective To investigate proliferative effects ofβ-NGF (Nerve growth factor) onhuman pterygium fibroblasts(HPF), and to analyse the pathogenesis mechanism ofpterygium.
Methods trkA and p75 expression was detected in HPF by immumofluorescencemethod. HPF were incubated with various concentrations ofβ-NGF. The efficacy ofβ-NGF on this cell line was assessed with MTT assay. Cell proliferation was evaluated bymeasurement of PCNA.
Results Expression of trkA and p75 was observed in HPF. Maximum stimulationoccurred at 48h for HPF. Incubation of HPF withβ-NGF resulted in a significant increasein PCNA compared with that of control cells.
Conclusion The findings demonstrate the potential proliferative effect ofβ-NGFbinding to trkA and p75 on HPF.
Objective To investigate the growth inhibition effects and the mechanisms of trkAinhibitor on human pterygium fibroblast.
Methods trkA and p75 expression was detected in HPF by immumofluorescencemethod. After being treated with 10～80nmol/L trkA inhibitor for 24～96h, the growthactivities of fibroblasts were studied by MTT colorimetry. Caspase-3 was inspected infibroblasts by spectrophotometric method. The apoptosis was detected by flow cytometery(FCM).
Results trkA inhibitor could effectively inhibit the in vitro growth of humanpterygium fibroblast in time and dose dependent manners. Caspase-3 expression wasincreased. The rates of apoptotsis were 8.26 %～29.62 % (P＜0.01).
Conclusion trkA inhibitor could induce apoptosis of human pterygium fibroblasts.Induicing apoptosis through changing the ratio of trkA and p75 in fibroblasts andup-reguating caspase-3 was probably one of its molecular mechanisms.
目的研究翼状胬肉组织中的NGF及其受体trkA、p75蛋白表达，探讨其与翼状胬肉形成的关系。
方法应用免疫组织化学检测30例翼状胬肉组织以及5例正常结膜组织中NGF、trkA和p75蛋白的表达。
结果NGF、trkA和p75蛋白在翼状胬肉组织的上皮细胞、成纤维细胞、血管内皮细胞中呈阳性表达；NGF、trkA蛋白在正常结膜组织上皮细胞和成纤维细胞中呈弱阳性表达；p75蛋白仅在正常结膜组织上皮细胞中呈弱阳性表达。
结论NGF、trkA、p75可能共同参与翼状胬肉的发生、发展过程。
第二部分p75和α-SMA在翼状胬肉组织的免疫定位
目的检测p75和α-SMA在翼状胬肉组织的表达定位。
方法正常结膜组织3例、翼状胬肉组织10例来自手术切除，3例正常角膜组织来自眼库。福尔马林浸泡并石蜡包被切片，用免疫组化方法检测trkA、p75和α-SMA在各种组织中的表达。利用共聚焦荧光显微镜通过免疫双标的方法检测p75和α-SMA在翼状胬肉组织及原代成纤维细胞中的定位。
结果p75、trkA和α-SMA在翼状胬肉组织成纤维细胞中表达明显。在翼状胬肉组织、正常角膜和结膜组织中检测到p75和trkA的表达。p75和α-SMA在翼状胬肉组织的头部可见阳性表达，在体部阳性表达较少。
结论在翼状胬肉组织头部p75和α-SMA表达在同一部位，p75表达上调可能参与细胞的增殖。
第三部分β-NGF对人翼状胬肉成纤维细胞增殖的影响
目的观察β-NGF(Nerve growth factor)对体外培养人翼状胬肉成纤维细胞(human pterygium fibroblasts，HPF)增殖的影响，探讨翼状胬肉发病机制。
方法免疫荧光法检测HPF中β-NGF受体trkA、p75的表达，MTT法检测不同浓度β-NGF对HPF的作用，Western-blot及RT-PCR法检测细胞核抗原(PCNA)来评估HPF增殖。结果trkA、p75在HPF中呈阳性表达；48h为β-NGF促增殖的高峰期；β-NGF在促HPF增殖的影响呈剂量依赖性。
结论β-NGF可能通过结合两受体trkA、p75促进了HPF的增殖。
第四部分trkA抑制剂对人翼状胬肉成纤维细胞凋亡的影响
目的探讨trkA抑制剂对体外培养人翼状胬肉成纤维细胞(HPF)生长抑制及其机制。
方法免疫荧光法检测HPF中β-NGF受体trkA、p75的表达，10～80nmol／LtrkA抑制剂作用24～96h后，MTF比色法检测细胞生长活性，分光光度法检测caspase-3在细胞中的表达，流式细胞仪测定细胞凋亡。
结果trkA抑制剂能显著抑制HPF的体外生长，呈剂量和时间依赖性；caspase-3活性检测表达增高，凋亡率为8．26％～29．62％(P＜0．01)。
结论trkA抑制剂可能通过改变HPF中trkA和p75的比率上调caspase-3诱导细胞凋亡。
Objective To investigate the expression of NGF, trkA and p75 in pterygium, and toanalyse their correlation with form of pterygium.
Methods NGF, trkA and p75 expression was detected in 30 pterygium tissues and 5normal conjunctiva tissues by immunohistochemical method.
Results Expression of NGF, trkA and p75 was significantly observed in epithelium,fibroblasts and vascular endothelial cells of pterygium tissues. Expression of NGF and trkAwas observed in epithelium and fibroblast of normal conjunctiva. Expression of p75 wasonly observed in epithelium of normal conjunctiva.
Conclusion NGF, trkA and p75 possibly participate in genesis and development ofpterygium.
Objective To determine localisation of p75 andα-SMA (α-smooth muscleactin) in pterygium tissues.
Methods Three normal conjunctiva and ten pterygium were surgically collected,and three normal cornea were collected from eye bank. Formalin fixed and paraffinembedded tissue sections were incubated with trkA, p75 polyclonal antibodies andα-SMA monoclonal antibody, and then examined by immunohistochemistry.Immunolocalisation of p75 andα-SMA was detected in pterygium and primarycultured fibroblasts by confocal microscopy.
Results Expression of p75,α-SMA and trkA was detected significantly infibroblasts of pterygium, p75 and trkA was examined in epithelium cells of pterygium,normal conjunctiva and cornea. In the pterygium head, p75 as well asα-SMA washeterogeneously expressed in cytoplasm of fibroblasts, whereas the expression was lessmarked in the body.
Conclusion It is suggested that p75 andα-SMA were concentrated in the sameposition of pterygium head, and p75 was possibly involved in cell proliferation.
Objective To investigate proliferative effects ofβ-NGF (Nerve growth factor) onhuman pterygium fibroblasts(HPF), and to analyse the pathogenesis mechanism ofpterygium.
Methods trkA and p75 expression was detected in HPF by immumofluorescencemethod. HPF were incubated with various concentrations ofβ-NGF. The efficacy ofβ-NGF on this cell line was assessed with MTT assay. Cell proliferation was evaluated bymeasurement of PCNA.
Results Expression of trkA and p75 was observed in HPF. Maximum stimulationoccurred at 48h for HPF. Incubation of HPF withβ-NGF resulted in a significant increasein PCNA compared with that of control cells.
Conclusion The findings demonstrate the potential proliferative effect ofβ-NGFbinding to trkA and p75 on HPF.
Objective To investigate the growth inhibition effects and the mechanisms of trkAinhibitor on human pterygium fibroblast.
Methods trkA and p75 expression was detected in HPF by immumofluorescencemethod. After being treated with 10～80nmol/L trkA inhibitor for 24～96h, the growthactivities of fibroblasts were studied by MTT colorimetry. Caspase-3 was inspected infibroblasts by spectrophotometric method. The apoptosis was detected by flow cytometery(FCM).
Results trkA inhibitor could effectively inhibit the in vitro growth of humanpterygium fibroblast in time and dose dependent manners. Caspase-3 expression wasincreased. The rates of apoptotsis were 8.26 %～29.62 % (P＜0.01).
Conclusion trkA inhibitor could induce apoptosis of human pterygium fibroblasts.Induicing apoptosis through changing the ratio of trkA and p75 in fibroblasts andup-reguating caspase-3 was probably one of its molecular mechanisms.
引文
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1．张明昌，王勇．重视翼状胬肉的基础与临床研究．中华眼科杂志．2007；43(10)868-871．
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5. Lambiase A, Micera A, Sgrulletta R, et al. Nerve growth factor and the immune system: old and new concepts in thecross-talk between immune and resident cells during pathophysiological conditions. Curr Opin Allergy Clin Immunol 2004;4:425-430.
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2．赵春明 张明昌 黄邦杏 张洁．NGF及其受体TRKA、P75在翼状胬肉组织中的表达．中国组织化学与细胞化学杂志，2008，17(6)：559-63．
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