目的：评估外用白介素(Interleukin, IL)-6特异性抑制剂托珠单抗滴眼液在调控角膜碱烧伤后修复的安全性和有效性。方法：6只角膜假烧伤小鼠局部使用托珠单抗滴眼(2.5 mg/mL)和6只角膜假烧伤小鼠局部使用生理盐水滴眼，分别作为实验组和空白组以评估托珠单抗滴眼液的安全性。30只碱烧伤小鼠，按照1∶1随机分配到治疗组和对照组，治疗组使用托珠单抗滴眼液滴眼，对照组使用生理盐水，每日6次，连续用14 d。通过前段光学相干断层扫描观察虹膜前粘连、角膜后弹力层脱离及角膜水肿，在体视显微镜下检查角膜瘢痕形成及上皮伤口愈合。在角膜切片上评估IL-6定位、肌成纤维细胞、免疫细胞浸润和角膜上皮化生。在角膜铺片上评估角膜新生血管和新生淋巴管面积。通过实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qRT-PCR)方法检测小鼠角膜 IL-6的表达水平。结果：对未进行碱烧伤的角膜使用托珠单抗治疗未观察到明显的角膜结构的损伤。角膜碱烧伤后可见角膜结构的破坏，角膜瘢痕形成并伴有角膜上皮伤口的延迟愈合。使用托珠单抗治疗后，虹膜前粘连的发生率从86.67%下降至20%(P <0.01)，角膜后弹力层脱离的发生率从93.33%下降至53.33%(P <0.05)，角膜厚度小于对照组[(100.03±15.73)μ m vs. (207.02±56.30)μ m，P＜0.001]，角膜混浊评分从对照组的3.76±0.44下降到治疗组的1.94±0.83(P <0.001)，治疗组在第5天(P <0.05)、第10天(P <0.001)和第14天(P <0.001)的上皮愈合率高于对照组。角膜碱烧伤后可见IL-6大量分布于角膜全层，且可见大量肌成纤维细胞形成及免疫细胞浸润，托珠单抗治疗后抑制了IL-6的表达(下降77.5%，P <0.05)，肌成纤维细胞数量从每视野(91.44±65.60)个减少至(12.89±10.51)个(P <0.01)，免疫细胞的数量从每视野(60.30±28.71)个细胞减少至每视野(6.80±3.82)个细胞(P <0.001)。此外，托珠单抗还减少角膜切片中每视野的杯状细胞数目由(11.3±5.29)个减少至(2.0±1.90个)(P <0.01)，并减少角膜新生血管和新生淋巴管的形成(分别减少了76.86%和71.16%，均P <0.001)。结论：局部使用托珠单抗抑制IL-6未见明显角膜毒性，且可以调控角膜碱烧伤后的修复。

Objective: To evaluate the safety and effect of topical IL-6 inhibitor tocilizumab eye drops in regulating corneal alkali burn repair. Methods: Six mice without corneal burns were locally treated with tocilizumab eye drops (2.5 mg/mL) and six mice with corneal pseudo burn were treated with saline, respectively, as experimental and blank groups to evaluate the safety of tocilizumab eye drops. 30 alkali burned mice were randomly divided into a treatment group and a control group in a 1:1 ratio. The treatment group received tocilizumab eye drops, while the control group received physiological saline solution 6 times per day for 14 days. Observe the anterior adhesion of the iris, detachment of the Descemet membrane, and corneal edema through anterior segment optical coherence tomography (AS-OCT), and examine corneal scarring and epithelial wound healing under a stereomicroscope. Evaluate IL-6 localization, myofibroblasts, immune cell infiltration, and corneal epithelial metaplasia on corneal sections. Evaluate corneal neovascularization and neovascularization area by whole-mount cornea staining. Detect the expression level of IL-6 in mouse cornea by qRT-PCR. Results: No significant damage to the corneal structure was observed in the treatment of unburned corneas with tocilizumab. After corneal alkali burns, the corneal structure was damaged, corneal scarring was formed, and delayed healing of corneal epithelial wounds was observed.After treatment with tocilizumab, the incidence of anterior synechia of the iris significantly decreased from 86.67% to 20% (P <0.01), the incidence of Descemet membrane detachment decreased from 93.33% to 53.33% (P <0.05), the corneal thickness was significantly less than that of the control group (100.03±15.73) μ m vs. (207.02±56.30)μ m (P <0.001), the corneal opacity score decreased from 3.76±0.44 in the control group to 1.94±0.83 in the treatment group (P <0.001), and the epithelial healing rate in the treatment group was significantly higher than that in the control group on day 5 (P <0.05), day 10 (P <0.001), and day 14 (P <0.001).After corneal alkali burns, IL-6 was distributed throughout the corneal layer, and a large number of myofibroblasts and immune cells were observed. After treatment with tocilizumab, the expression of IL-6 was inhibited (decreased by 77.5%, P <0.05), the number of myofibroblasts decreased from (91.44±65.60) per field to (12.89±10.51) per field (P <0.01), and the number of immune cells decreased from (60.30±28.71) cells per field to (6.80±3.82) cells per field (P <0.001). In addition, tocilizumab also reduced the number of goblet cells per field in corneal sections (from 11.3±5.29 to 2.0±1.90) (P <0.01), and reduced the formation of corneal neovascularization and neovascular lymphatic vessels (by 76.86% and 71.16%, respectively, both P <0.001). Conclusion: Topical use of tocilizumab to inhibit IL-6 showed no significant corneal toxicity and can regulate the repair of cornea after alkali burns.

目的: 研究 TGF-β1 短期眼部应用对兔角膜碱烧伤后整合素 β1 表达和角膜上皮愈合的影响,探求其对角膜碱烧伤的治疗作用。

方法: 制备大耳白家兔角膜碱烧伤模型, 一组给予 TGF-β1 (浓度为 200 ng /ml) 局部滴眼, 每日 3 次, 连续 7 日; 另一组给予 PBS 溶液代替, 处理相同。于角膜碱烧伤后每日观察角膜上皮愈合面积, 并于烧伤后 6 h、1 d、3 d、7 d 和 14 d 5 个时间点应用免疫组化方法检测 TGF-β1 实验组与 PBS 组角膜整合素 β1 表达情况。

结果: 烧伤后 4 d、10 d、11 d、12 d 和 14 d 实验组和对照组上皮愈合率比较, 差异有统计学意义(P < 0.05) , 两组随着上皮修复过程的进行, 整合素 β1 的表达均逐渐增加, 烧伤后 7 d、14 d两个时间点实验组和对照组整合素 β1 平均灰度值比较, 差异有显著性(P < 0.05) 。

结论: TGF-β1 在活体实验中能促进整合素 β1 的表达, 而后者的增加可以促进角膜上皮细胞向损伤区域的移行和粘附, 从而减少碱烧伤愈合过程中上皮再次脱落现象, 有利于创伤愈合。

Purpose: To observe the effect of TGF-β1 applied topically to the alkali-injured rabbit eye on corneal epithelial wound healing and expression of integrin β1 and its therapeutic action on corneal alkali burns.
Methods: Alkali burn was produced in 60 corneas from 30 rabbits. Two groups were randomly divided. One group was treated with TGF-β1 solution (200 ng /ml) topically 3 times one day within the first 7 days, the other group was treated with phosphate-buffered saline (PBS) solution. The injured eyes were photographed after the fluorescence staining with a digital camera and the pictures were analyzed with computer-aided picture analysis system to calculate the rate of corneal epithelial healing. The expression of integrin β1 was investigated in the point 6 h, 1 d, 3 d, 7 d, 14 d after the injury by means of immunohistochemical analysis.
Results: On the 4th, 10th, 11st, 12nd and 14th days after the burning, the rate of corneal epithelial healing of TGF-β1 groups was markedly higher than that of the PBS group (P < 0.05) . The expression of integrin β1 in the cornea epithelial cells gradually increased during the wound healing. On the 7th and 14th days after the burning, the expression of integrin β1 in the cornea epithelial cells of TGF-β1 group was remarkably  higher than that of the PBS group(P < 0.05) .

Conclusions: TGF-β1 could up-regulate integrin β1 in vivo corneal alkali burn model, which could stimulate the cornea epithelial cells to migrate and adhere to the cornea stroma, that can reduce the cases of the epithelial cells_detachment from the cornea stroma and sustain the corneal reepithelization.