角膜上皮干细胞缺失动物模型的方法研究

Research of corneal epithelial stem cell deficiency animal models

目的:建立兔角膜上皮干细胞缺失动物模型,为人工生物角膜上皮实验研究奠定基础。方法:方法一,单纯去除角膜缘浅板层环周内外宽各2mm,深约100～150μm角膜组织;方法二,在方法一的基础上,再去除角膜中央浅板层组织约100～150μm。术后,裂隙灯显微镜下观察,以角膜混浊、上皮荧光素染色及新生血管为参数评价,3项参数得分之和为该动物时间点总评分。以角膜新生血管评分在3分以上,角膜上皮混浊在2分以上,采用印迹细胞法角膜面取材行角蛋白K3单克隆抗体间接免疫荧光法检测表达为阴性的。结果:单纯去除角膜缘的动物模型10眼有4眼成功(40%),从制作至成功平均时间29.5±1.3d。角膜缘联合角膜中央板层组织共同去除组,10眼均成功(100%),从制作至成功平均时间为15.8±0.8d,两组成功率对比差异有统计学意义(χ2=8.58,P<0.01);成功天数比较差异有统计学意义(t=24.5,P<0.01)。两组成功模型角膜采用印迹细胞法取材行角蛋白K3单克隆抗体间接免疫荧光法检测表达均为阴性。结论:采用角膜缘联合角膜中央上皮共同去除法制备角膜上皮干细胞缺乏模型成功率高,周期短,值得推广应用。

AIM： To establish the rabbit corneal epithelial stem cell deficiency animal models and basis of application of artificial biological corneal epithelial. METHODS： Method 1 ：the superficial layers were removed, ranged from 2ram inner to 2mm outer of limbus cornea 100-1501Jm deep; Method 2：on the basis of Method 1, the superficial layers of central corneas were removed 100- 150μm deep. The cloudiness of cornea, epithelium fluorescein staining and new vessels as parameters were observed by slit lamp microscope after operation. The sum of the three parameters was the total score at the corresponding time point. The corneal samples, whose scores of new vessels were over 3 and scores of cloudiness were over 2, were drawn by imprinting method, and the results of the detection of the keratin 3 on corneal samples by indirect immunofluorescence were negative. RESULTS： Four in 10 eyes（40%） were successful by method 1, and the forming meantime was 29.5 ± 1.3 days; all the 10 eyes（100%） were successful by method 2, and the forming meantime was 15.8 ± 0.8 days. There were statistically significant differences in successful ratio （χ^2 = 8.58,P〈 0.01） and the meantime（t= 24. 5, P〈 0. 01） between the two methods. The corneas from successful models were drawn by imprinting method, and the results of the detection of the keratin 3 by indirect immunofluorescence were negative. CONCLUSION： To establish the corneal epithelial stem cell deficiency animal models by removing the Iimbus and central corneal epithelia took shorter meantime with higher successful ratio.