β-NGF对人翼状胬肉成纤维细胞增生的影响  

The effects of β-NGF on proliferation of human pterygium fibroblasts

作　　者：赵春明[1] 张明昌[1] 晏雪莹[1] 毛晓春[2] 

机构地区：[1]华中科技大学同济医学院附属协和医院眼科,武汉430022 [2]华中科技大学同济医学院附属同济医院眼科,武汉430032

出　　处：《眼科研究》2009年第11期955-959,共5页Chinese Ophthalmic Research

摘　　要：目的观察神经生长因子β(β-NGF)对体外培养的人翼状胬肉成纤维细胞(HPF)增生的影响,探讨翼状胬肉的发病机制。方法对翼状胬肉组织标本行组织块培养。待细胞生长汇合至80%后以0.25%胰蛋白酶+0.02%EDTA1∶1混合消化细胞,取3～5代细胞用于实验。通过细胞中波形蛋白、角蛋白及α平滑肌肌动蛋白的表达鉴定培养的细胞,免疫荧光法检测HPF中β-NGF受体trkA、p75的表达,MTT法检测不同质量浓度β-NGF对HPF的作用,Westernblot及RT-PCR法半定量检测细胞核抗原(PCNA)来评估HPF的增生。结果波形蛋白、α平滑肌肌动蛋白、trkA和p75在HPF中呈阳性表达;MTT结果显示,在β-NGF作用12、24、48、72和96h后,48h为β-NGF促增生的高峰期;在不同质量浓度β-NGF(5～50ng/mL)作用下,HPF中PCNA蛋白及mRNA的表达与0和1ng/mLβ-NGF作用组之间比较差异均有统计学意义(Fprotein=24.980,P=0.000;FmRNA=64.490,P=0.000)。结论NGF可能通过结合两受体trkA、p75促进了HPF的增生。Objective Our previous research demonstrated that trkA and p75 receptors of nerve growth factor（β-NGF） are expressed in human pterygium fibroblasts（HPF） ,and trkA is expressed only in eonjunetiva. The purpose of present study was to investigate the effects of β-NGF on proliferation of HPF and analyse the pathogenesis mechanism of pterygium. Methods The HPF specimen was obtained from Union Hospital of Tongji Medical College, Huazhong University of Science and Technology during the surgery. Explant culture technique was used for the primary culture of HPF tissue. The cells of confluenting 80% were collected and digested using 0. 25% tripsin ＋ 0. 02% EDTA（ 1 ： 1 ） and the third to fifth generation of cells were utilized in the experiment. Different concentrations of β-NGF was added in medium. Cultured cells were identified using vimentin, keratin and α-SMA. MTT was used to determine the proliferation of HPF after addition of β-NGF. The expression of trkA and p75 in HPF was detected by immumofluorescence method. Cell proliferation also was semi-quantitatively analyzed by detect of expressions of PCNA protein and mRNA in HPF using Western blot and RT-PCR. Results Cultured HPF cells showed the positive responses for vimentin,α-SMA,trkA and p75 but absent reaction for keratin. MTT revealed that the OD value of HPF cells was gradually enhanced with the increase of β-NGF concentration in 12,24,48,72 and 96 hours after 13-NGF action with the maximum stimulation at 48 hours. The expression of PCNA protein and mRNA in HPF was significantly different among various concentrations of β-NGF groups （ Fprotei, = 24. 980, P = 0. 000 ; FmRNA = 64. 490, P = 0. 000 ） and increased from 5 ng/mL β-NGF group through 50 ng/mL β-NGF group in comparison with 0 and 1 ng/mL β-NGF group（P 〈 O. 05）. Conclusion The findings demonstrate the potential proliferative effect of β-NGF binding to trkA and p75 on HPF.

关 键 词：翼状胬肉 成纤维细胞 Β-神经生长因子 TRKA P75 神经生长因子受体 

分 类 号：R777.33[医药卫生—眼科]