Efficacy and safety of mitomycin C as an agent to treat corneal scarring in horses using an in vitro model.
Efficacy and safety of mitomycin C as an agent to treat corneal scarring in horses using an in vitro model.
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DOI：
10.1111/j.1463-5224.2010.00782.x
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发表时间：
2010-07
影响因子：
1.6
通讯作者：
Mohan RR
中科院分区：
文献类型：
--
作者：
Buss DG;Sharma A;Giuliano EA;Mohan RR
丝裂霉素C（MMC）在临床上用于治疗人类患者的角膜瘢痕形成。我们通过使用体外模型检查MMC在疾病早期和晚期的作用，研究了MMC治疗马角膜瘢痕的安全性和有效性。使用开发的马角膜成纤维细胞（ECF）的体外模型。在无血清条件下，在存在或不存在转化生长因子β 1（TGFβ1）的情况下，通过培养原代ECF产生马角膜成纤维细胞或肌成纤维细胞培养物。在将MMC应用于培养物一次2分钟后，通过剂量依赖性台盼蓝拒染和MTT [（3- 4，5-二甲基噻唑-2-基）-2，5-二苯基溴化四氮唑]测定确定马角膜的MMC剂量。通过Western印迹、免疫细胞化学和/或定量实时聚合酶链反应测定角膜瘢痕标记物（α-平滑肌肌动蛋白和F-actin）的mRNA和蛋白表达，确定MMC控制马角膜瘢痕形成的疗效。0.02%或更低MMC单次2分钟处理未改变ECF表型、活力或细胞增殖，而0.05%或更高MMC剂量显示轻度至中度细胞毒性。在无血清条件下，1 ng/ml的TGFβ1在ECF中显示显著的肌成纤维细胞形成。在TGFβ1刺激后24小时（早期）单次2分钟，0.02%MMC处理显著降低了ECF向肌成纤维细胞的转化，然而，在TGFβ1刺激后11天单次0.02%MMC处理显示出中度肌成纤维细胞抑制。MMC通过降低角膜成纤维细胞向肌成纤维细胞的转分化程度，安全有效地减少ECF中的瘢痕形成。需要在马中使用MMC进行进一步的临床体内研究。
Mitomycin C (MMC) is used clinically to treat corneal scarring in human patients. We investigated the safety and efficacy of MMC to treat corneal scarring in horses by examining its effects at the early and late stages of disease using an in-vitro model. An in-vitro model of equine corneal fibroblast (ECF) developed was used. The equine corneal fibroblast or myofibroblast cultures were produced by growing primary ECF in the presence or absence of transforming growth factor beta-1 (TGFβ1) under serum-free conditions. The MMC dose for the equine cornea was defined with dose-dependent trypan blue exclusion and MTT [(3-4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays after applying MMC to the cultures once for 2 minutes. The efficacy of MMC to control corneal scarring in horses was determined by measuring mRNA and protein expression of corneal scarring markers (α-smooth muscle actin and F-actin) with western blotting, immunocytochemistry and/or quantitative real-time polymerase chain reactions. A single 2 minutes treatment of 0.02% or less MMC did not alter ECF phenotype, viability, or cellular proliferation whereas 0.05% or higher MMC doses showed mild-to-moderate cellular toxicity. The TGFβ1 at 1ng/ml showed significant myofibroblast formation in ECF under serum-free conditions. A single 2 minute, 0.02% MMC treatment 24 hours (early) after TGFβ1 stimulation significantly reduced conversion of ECF to myofibroblasts, however, a single 0.02% MMC treatment 11 days after TGFβ1 stimulation showed moderate myofibroblast inhibition. That MMC safely and effectively reduced scarring in ECF by reducing the degree of transdifferentiation of corneal fibroblasts to myofibroblasts in vitro. Further clinical in-vivo investigations are warranted using MMC in horses.
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影响因子：
3.4
作者：
Mohan, Rajiv R.;Stapleton, W. Michael;Wilson, Steven E.
通讯作者：
Wilson, Steven E.
影响因子：
2.8
作者：
Carones, F;Vigo, L;Vacchini, L
通讯作者：
Vacchini, L
影响因子：
1.1
作者：
Rayner, SG;Van Zyl, N
通讯作者：
Van Zyl, N
影响因子：
3.4
作者：
Mohan, RR;Hutcheon, AEK;Wilson, SE
通讯作者：
Wilson, SE
影响因子：
1.6
作者：
Buss DG;Giuliano EA;Sharma A;Mohan RR
通讯作者：
Mohan RR