A comparative study on the inhibitory effects of commonly used clinical drugs on in vitro fibroblasts from recurrent pterygium

Authors: Sun Yajie,  Lin Tingting,  He Yanjin,  Zhu Limin,  Gao Yang,  Zhou Bo

DOI: 10.3760/cma.j.issn.2095-0160.2017.06.006
Published 2017-06-10
Cite as Chin J Exp Ophthalmol, 2017,35(06): 512-519.

Abstract

Background

Pterygium is one of the common ocular surface disorders, and the main drugs for pterygium include dexamethasone (DXM), interferon α-2b (IFN-α2b), mitomycin C (MMC), 5-fluorouracil (5-FU), cyclosporin A (CsA) and tacrolimus (FK506). However, the efficacy of these drugs on the fibroblasts from recurrence pterygium is unelucidated.

Objective

This study was to compare the efficacy of DXM, IFN-α2b, MMC, 5-FU, CsA and FK506 on proliferation and apoptosis of recurrent pterygium-derived fibroblasts in vitro.

Methods

The specimens of recurrence pterygium were collected during surgery in Tianjin Medical University Ophthalmological Hospital from May 2015 to July 2016 under the written informed consent.Fibroblasts were isolated and cultured by explant culture method and identified by immunochemistry.DXM, IFN-α2b, MMC, 5-FU, CsA and FK506 were added into the medium for 48 hours, respectively, and the cells cultured without drug were used as the control group.The inhibitory efficiency of different concentrations of DXM, IFN-α2b, MMC, 5-FU, CsA and FK506 on the cell proliferation was assayed by cell counting kit-8 (CCK-8), and 50% inhibiting concentration (IC50) of the drugs was calculated.The cells were treated by the IC50 dose of drugs for 48 hours, and cell apoptotic proportion and cell cycle were assessed by flow cytometry analysis.The expression of proliferating cell nuclear antigen (PCNA) in the cells after treated by drugs was detected by immunochemistry.

Results

Cultured cells grew well with the fusiform shape and radial arrangement.Vimentine showed the positive expression and keratin was absently expressed in the cells.The IC50 to the cells was (3.5×103±2.83×10-2)mg/L, (6.1×102±3.6×10-3)mg/L, (3.2×10-1±1×10-4)mg/L, (2.2×101±1.2×10-3)mg/L, (6.3×101±2.5×10-3)mg/L and (6.0×101± 0.0×100) mg/L in the DXM, IFN-α2b, MMC, 5-FU, CsA and FK506, respectively.In the 48 hours after treated by the IC50 drugs, the apoptotic ratio was (35.00±3.21)%, (30.37±1.67)%, (26.11±0.75)%, (22.01±0.07)%, (20.95±1.68)% and (19.85±0.52)% in the IFN-α2b group, CsA group, MMC group, FK506 group, DXM group and 5-FU group, which was significantly higher than (11.38±2.18) % in the control group (all at P<0.05). The cell proportion of G0/G1 phase, S phase and G2/M phase was (85.64±2.62)%, (5.29±1.56)% and (2.73±2.66)% in the control group, and the cell proportion of G0/G1 phase was reduced, while that of S phase or G2/M phase was considerably increased in various drug groups (all at P<0.05), with the blocking efficiency of cell cycle was in turn MMC, CsA, 5-FU, DXM, IFN-α2b and FK506.The expressional rate of PCNA in the cells was (95.00±2.00)%, (82.67±5.04)%, (80.00±2.78)%, (64.00±6.55)%, (38.00±3.00)%, (32.00±4.36)% and (29.67±3.02)% in the control group, FK506 group, DXM group, 5-FU group, IFN-α2b group, CsA group and MMC group, showing a significant difference among the groups (F=25.995, P<0.01), and the expressional rate of PCNA was significant lower in various drug groups than that in the control group (all at P<0.05).

Conclusions

DXM, IFN-α2b, MMC, 5-FU, CsA and FK506 are all able to inhibit the proliferation and promote the apoptosis of recurrent pterygium-derived fibroblasts in vitro, and MMC and CsA appear to have a stronger effect.

Key words:

Pterygium/surgery; Pterygium/drug therapy; Recurrence; Fibroblasts; Dexamethasone; Interferon-α; Mitomycin C; 5-Fluorouracil; Cyclosporin A; Tacrolimus; Cells, cultured

Contributor Information

Sun Yajie
Tianjin Medical University Ophthalmological Hospital, Tianjin 300000, China
Lin Tingting
He Yanjin
Zhu Limin
Gao Yang
Zhou Bo
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Updated: September 4, 2019 — 1:08 pm