环孢素A抑制结膜干燥症鼠结膜上皮细胞凋亡的研究

Inhibition of cyclosporine on apoptosis of conjunctival epithelium in experimental murine xerophthalamia

目的探讨环孢素A(CsA)是否抑制结膜干燥症实验鼠模型的结膜上皮细胞的凋亡。方法30只C57BL6小鼠皮下注射东莨菪碱12d制造结膜干燥症模型鼠,各取10只为干眼对照组、干眼+CsA组和干眼+载体组。另取10只为正常组,作为未处理的对照。采用PAS染色及免疫组织化学方法,对小鼠结膜上皮细胞中的杯状细胞及caspase-3、Bax和bcl-2等相关基因蛋白的表达进行检测。结果正常组和干眼+CsA组结膜的上皮杯状细胞的数目明显高于干眼对照组和干眼+载体组。干眼对照组和干眼+载体组结膜上皮细胞中caspase-3及Bax阳性表达的细胞数均明显高于正常组和干眼+CsA组,与杯状细胞数目成负相关;而bcl-2阳性表达则相反。结论CsA治疗结膜干燥症的主要机制在于抑制细胞凋亡。

Objective This study was designed to determine if topical cyclosporine （CsA） treatment inhibits apoptosis of conjunctiva epithelial cell in a murine model of xerophthalamia. Methods Dry eye model of C57BL6 mice was induced by subcutaneous injection of scopolamine TID and exposure to an air draft and low-humidity environment for 16 hours per day for 12 days. The 30 mice were randomly assigned to dry eye control group without topical treatment, dry eye ＋ CsA group（ topical administration of 1μL 0.05% CsA after injection of TID ） and dry eye ＋ vehicle group（ topical administration of 1μL of the castor oil vehicle of CsA after injection of TID）. Normal mice served as controls. Experimental mice were killed 12 clays later, and conjunctival goblet cell number was counted with period acid Schiff （PAS） staining. The conjunctiva epithelium was examined by immunohistochemical staining to detect the goblet cells and the expression of caspase-3,bax and bcl-2. ResuIts The number of conjunctival epithelial goblet cells was more in normal controls（ 52.3 ± 2.3 ） and dry eye ＋ CsA groups（ 50. 8 ± 2.8 ） than dry eye control and dry eye ＋ vehicle groups（27.3±2.3 and 25.0 ± 1.8） （P 〈0.01 ,P 〈0.01 ）. Expressions of caspas-3 and bax in conjunctival epithelium cells were increased in dry eye control and dry eye ＋ vehicle groups compared with normal and dry eye ＋ CsA groups respectively（ 401.67 ± 101.59,392.27± 93.58,303.35 ± 90. 45,289 ±73.82 vs 73.52 ± 36.58,69. 94 ± 33.68, 52.47 ±27.3,56. 39 ± 21.38） （ P 〈 0.01 ） ; the expresson of bcl-2 was decreased in the experiment groups compared with matched control（ 77.49±35.69,82.49 ±39.57 vs 253. 18 ±56.39,247.35 ±27.37） （P 〈0.01,P 〈 0.01）. There was a significant positive correlation between goblet cell number and the cell number expressing bcl-2, however a negative correlation between goblet cells and caspase-3 or bax expression was demonstrated. ConcIusion Topical CsA significantly protects conjunctival epithelial from apoptosis in experimental murine xerophathalamia. Inhibition of apoptosis appears to be a key mechanism for the therapeutic effect of CsA on xerophthalamia.