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 (FKS06).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-o2b,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.%背景 翼状胬肉是眼科常见的眼表疾病,临床上常用的防治翼状胬肉复发的药物包括地塞米松(DXM)、干扰素α-2b(IFN-α2b)、丝裂霉素C(MMC)、5-氟尿嘧啶(5-FU)、环孢素A(CsA)和他克莫司(FK506),但鲜见关于这6种药物对复发性翼状胬肉成纤维细胞生物学行为抑制作用的比较研究. 目的 观察DXM、IFN-α2b、MMC、5-FU、CSA和FK506对体外培养的人复发性翼状胬肉成纤维细胞的抑制作用,筛选治疗复发性翼状胬肉的最佳药物. 方法 收集2015年5月至2016年7月在天津医科大学眼科医院行复发性翼状胬肉切除术的患者术中切除的组织标本7例7眼,采用组织块培养法分离和培养成纤维细胞,采用免疫组织化学法对培养细胞进行鉴定.分别在培养液中添加DXM、IFN-α2b、MMC、5-FU、CsA和FK506作用48 h,未添加药物的培养细胞作为对照组.采用CCK-8法检测各种药物对培养细胞的生长抑制率,分别计算药物的半数抑制浓度(IC50).在药物IC50剂量下作用48 h,采用流式细胞仪检测各组细胞凋亡比例和细胞周期的变化;采用免疫组织化学法检测细胞中增生细胞核抗原(PCNA)的表达变化. 结果 培养的细胞为长梭形,呈旋涡状排列,细胞中波形蛋白表达阳性,角蛋白无表达.DXM、IFN-α2b、MMC、5-FU、CsA和FK506对成纤维细胞的IC50分别为(3.5×103±2.83×10-2)、(6.1×102±3.6×10-3)、(3.2×10-1±1×10-4)、(2.2×101±1.2× 10-3)、(6.3×101±2.5×10-3)和(6.0×101±0.0×100) mg/L.在IC50剂量下分别作用后48 h,IFN-α2b组、CsA组、MMC组、FK506组、DXM组和5-FU组凋亡细胞比例分别为(35.00±3.21)％、(30.37±1.67)％、(26.11±0.75)％、(22.01±0.07)％、(20.95±1.68)％和(19.85±0.52)％,与对照组的(11.38±2.18)％比较细胞凋亡比例均明显升高,差异均有统计学意义(均P＜0.05).对照组G0/G1期、S期和G2/M期细胞比例分别为(85.64±2.62)％、(5.29±1.56)％和(2.73±2.66)％,各药物组G0/G1期细胞比例明显下降,S期和G2/M期细胞明显升高,差异均有统计学意义(均P＜0.05),对细胞周期阻滞效果由强到弱依次为MMC、CsA、5-FU、DXM、IFN-α2b和FK506.对照组细胞中可见有大量PCNA阳性表达,各药物组细胞中PCNA表达强度均弱于对照组.对照组、FK506组、DXM组、5-FU组、IFN-α2b组、CsA组和MMC组细胞中PCNA的阳性表达率分别为(95.00±2.00)％、(82.67±5.04)％、(80.00±2.78)％、(64.00±6.55)％、(38.00±3.00)％、(32.00±4.36)％和(29.67±3.02)％,总体比较差异有统计学意义(F=25.995,P=0.000),各药物组细胞中PCNA阳性表达率均明显低于对照组,差异均有统计学意义(均P＜0.05).结论 DXM、IFN-α2b、MMC、5-FU、CsA和FK506均可抑制复发性翼状胬肉成纤维细胞的增生并促进细胞凋亡,其中MMC和CsA的作用更为显著.
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