背景翼状胬肉复发是翼状胬肉手术切除的常见并发症,其发生机制与细胞增生、炎症过程和新生血管形成有关,用抗增生药物控制翼状胬肉复发是目前研究的热点和方向.研究证实罗格列酮具有显著的抗炎、抗肿瘤、抑制新生血管作用,但其对翼状胬肉复发的作用研究较少.目的观察过氧化物酶体增生物激活受体(PPARγ)激动剂罗格列酮对体外培养的人翼状胬肉成纤维细胞(HPFs)增生和凋亡的影响,寻求预防和辅助治疗翼状胬肉术后复发的新药物.方法对手术切除的原发性进展期翼状胬肉组织采用组织块培养法进行原代和传代培养,用酶消化法对培养的HPFs进行人工纯化.HPFs培养板中加入终浓度为5、10、25、50、75、100、150、200、400 μmol/L的PPARγ激动剂罗格列酮,同时设未加药物干预的对照组和只加培养液的空白对照组.观察不同浓度罗格列酮作用12-72 h后对HPFs生长的影响,MTT比色法检测细胞的生长抑制率,流式细胞仪检测细胞周期时相和凋亡的变化.实时定量聚合酶链反应(real-time PCR)检测增生细胞核抗原(PCNA)的表达变化.结果培养的HPFs呈长梭形,体积较大,呈铺路石样排列,对波形蛋白呈阳性反应,角蛋白反应阴性.罗格列酮作用后,培养的HPFs形态变圆.色变淡,核固缩或碎裂,局部细胞膜不完整.25～125μmoL/L罗格列酮作用12-72 h可抑制HPFs的增生,其作用呈剂量和时间依赖性(剂量:F=158.312,P=0.006;时间:F=1.924,P=0.135).流式细胞仪检测结果显示,25、75、125μmol/L罗格列酮作用于HPFs 24 h,随着浓度的增加G0/G1期细胞百分比逐渐上升,S期细胞百分比逐渐下降(P＜0.05),表明细胞阻滞于G0/G1期.罗格列酮组HPFs凋亡率明显高于对照组(P＜0.05),且随罗格列酮浓度的升高、作用时间的延长、HPFs凋亡率上升,差异有统计学意义(P＜0.05).随着罗格列酮浓度的增加,PCNA mRNA在HPFs中的表达明显降低,差异有统计学意义(F=3244.329,P＜0.05).结论 PPARγ激动剂以剂量和时间依赖的方式抑制HPFs的增生,并诱导其凋亡.大剂量的PPARγ激动剂可抑制细胞PCNA的合成和表达.%Background Recurrence of pterygium is a common complication after the surgical excision of pterygium,and this procedure is related to cell proliferation,inflammation and neovascularization.Researches determined that rosiglitazone can suppress inflammation and neovaseularization and inhibit proliferation,hut few studies concerning the effect of rosiglitazone on pterygium were performed. Objective The aim of this study was to investigate the effect of peroxisome proliferator-activated receptor γ agonist on the proliferation and apoptosis of human pterygium fibroblasts(HPFs)in culture and search for a new drug to prevent and cure the recurrence after pterygium surgery. Methods Human pterygium samples were obtained during surgery and HPFs were cultured and purified using an explant method and 0.25%trypsin digestion,respectively.The identity of cultured HPFs was confirmed by immunohistochemistry using anti-vimentin and keratin antibodies.Rosiglitazone with the concentrations of 0(control),5,10,25,50,75,100,150,200,400μmol/L was then added in the culture medium for 12,24 or 72 hours.1%DMSO was used as blank control.The MTT method was used to assay the biologic effects of rosiglitazone on HPFs.Cell cycle distribution and apoptosis of HPFs after rosiglitazone treatment were studied by flow cytometic analysis.The expression of proliferating cell nuclear antigen(PCNA)mRNA in HPFs was detected by real-time PCR. Result Cultured HPFs radially migrated outward from the pterygium block,and then grew in long fusiform shape,showing positive response for vimentin and negative for keratin.The HPFs became round and thin with loose distribution after the addition of rosiglitazone.Following 25-125 μmol/L rosiglitazone administration for 12,48 or 72 hours,the A490 value of HPFs significantly declined with the increase of dosage(F=158.312,P=0.006)and lapse of time(F=1.924,P=0.135).Following the treatment of 25,75 or 125 μmol/L rosiglitazone for 24 hours,the number of HPFs in G0/G1 phase was markedly elevated;while the cell numbers in S phase decreased significantly in comparison with the control group(P＜0.05).The apoptotic rate of HPFs in the 25,75 and 125 μmol/L rosiglitazone groups significantly increased with the increase of rosiglitazone concentration(P＜0.05).Real-time PCR revealed that after 24 hours of rosiglitazone treatment,the expression of PCNA mRNA in HPFs was suppressed in a dose-dependent manner(F=3244.329,P＜0.05). Conclusion Rosiglitazone inhibits HPFs proliferation,arrests their cell cycle progression in G0/G1 phase,induces apoptosis of HPFs and depresses the synthesis of PCNA in a dose-and time-dependent manner.
展开▼
