昼夜节律颠倒对小鼠角膜上皮创伤修复的影响

Wound healing of corneal epithelium in mice affected by reversed circadian rhythms

目的探究昼夜节律颠倒对角膜上皮创伤修复的影响。方法选取6~8周龄C57BL/6雄性小鼠90只,按照随机数字表法分为昼夜节律正常(LD)组和昼夜节律颠倒(DL)组。置于昼夜节律箱中饲养12d,在小鼠角膜中央标记直径为2mm的圆形区域,用高尔夫样刀刮除标记区域上皮层,分别使用荧光素钠、苏木精-伊红染色观察并记录角膜再上皮化过程。使用DAPI、FITC标记抗鼠Ly6G抗体和PE标记抗鼠γδTCR抗体进行免疫荧光染色,每隔6h观察角膜创伤后细胞分裂、中性粒细胞和γδT细胞数量的动态变化,直至42h。结果角膜上皮创伤后0、6、12、18和24hLD组角膜上皮缺损面积百分比分别为(100.000±0.000)%、(37.677±5.243)%、(14.959±1.739)%和(0.000±0.000)%,DL组角膜上皮缺损面积百分比分别为(100.000±0.000)%、(10.967±1.065)%、(1.985±0.106)%和(0.000±0.000)%,总体比较差异有统计学意义(F=213.718,P=0.000)。角膜上皮创伤前,DL组和LD组角膜上皮层厚度分别为(33.983±1.074)μm和(33.993±0.904)μm,差异无统计学意义(P>0.05);角膜上皮损伤后24h,DL组角膜上皮厚度为(19.473±0.856)μm,明显大于LD组(17.485±0.718)μm,差异有统计学意义(P<0.05)。苏木精-伊红染色显示,创伤24h后2个组小鼠角膜上皮细胞皆排列疏松、紊乱、形态不规则。LD组角膜上皮主要为基底细胞,DL组除了基底细胞外,还有少量扁平细胞。角膜上皮创伤后LD组和DL组上皮分裂细胞数量、中性粒细胞数量和γδT细胞数量总体比较,差异均有统计学意义(P<0.05)。结论昼夜节律颠倒可通过促进角膜上皮分裂细胞、中性粒细胞和γδT细胞募集高峰期提前,进而调节角膜上皮创伤修复过程。

Objective To observe the effect of reversed circadian rhythms on wound healing of mouse corneal epithelium. Methods Ninety male C57BL/6 mice were divided into LD group (12 hours light/12 hours dark) and DL group (12 hours dark/12 hours light) by random number table,and then were placed in circadian rhythm box for 12 days.The circular area was scarped and marked as 2 mm diameter area in the center of the mouse's cornea with a golf-like knife.The dynamics of epithelial healing in the wound area were observed under microscope by fluorescein staining and hematoxylin-eosin staining.Besides,being marked antibodies of anti-Ly6G-FITC,anti-γδT-PE and DAPI,dynamic changes of the dividing cells,neutrophils and γδT cells were also investigated for every 6 hours until 42 hours.All mice were treated in accordance with the Association for Research in Vision and Ophthalmology's Statement for the Use of Animals in Ophthalmology and Vision Research and the guidelines of the Animal Experimental Committee at Jinan University (JN-A-2002-01). Results In LD group,percentage of corneal epithelial defective area were (100.000±0.000)%,(37.677±5.243)%,(14.959±1.739)% and (0.000±0.000)% after wounding 0 hour,6,12,18 and 24 hours.In DL group,percentage of the corneal epithelial defective area were (100.000±0.000)%,(10.967±1.065%)%,(1.985±0.106)% and (0.000±0.000)% after wounding 0 hour,6,12,18 and 24 hours.The healing rate in DL group was higher than that in LD group,with a significant difference between them ( P <0.05).As with the uninjured corneal,thickness of corneal epithelium was (33.983±1.074)μm in DL group and (33.993±0.904)μm LD group,with no statistically significant difference between them ( P >0.05).After 24 hours,thickness of corneal epithelium in DL group was (19.473±0.856)μm,and was more than that in LD group [(17.485±0.718)μm],with a significant difference between them ( P <0.05).Paraffin section of wounded corneal epithelium after 24 hours by hematoxylin and eosin staining showed that corneal epithelium cells arranged loosely and disorderly and were in irregular shape in both groups.The epithelium were mainly basal cells in LD group,while epithelium included basal cell and a few pinacocytes in DL group.After corneal epithelium wounded,the number of cell division,neutrophils and corneal limbus γδT cells in two groups were statistically significant difference,respectively( P <0.05). Conclusions Reversed circadian rhythms can significantly regulate the wound healing of corneal epithelium.