ABSTRACT
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 conjunctiva. 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 β-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(F_(protein) = 24. 980, P = 0. 000; F_(mRNA) = 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 ＜ 0. 05) . Conclusion The findings demonstrate the potential proliferative effect of β-NGF binding to trkA and p75 on HPF.