高渗透压对兔眼表上皮和黏蛋白5AC表达的影响

目的 探讨高渗透压对兔眼表上皮和黏蛋白(MUC)5AC表达的影响.方法 实验研究.将18只健康新西兰大白兔采用计算机随机数字法分为3组,分别为高渗盐溶液(500 mmol/L)、生理盐水(308 mmol/L)滴眼组及空白对照组,每组6只兔.实验第0、7、14天测定泪液分泌量与泪膜破裂时间(BUT),结膜印迹细胞学测定杯状细胞密度.实验第7、14天处死动物,行角结膜光镜和电镜观察,结膜组织末端脱氧核苷酸转移酶介导的脱脲苷三磷酸缺口末端标记法(TUNEL)检测,第14天的结膜组织另行MUC5AC表达的免疫组织化学染色和免疫印迹法检测.同一时间点上泪液分泌量值、BUT值和杯状细胞密度的组间比较采用单因素方差分析法,进一步两两比较采用Bonferroni法;若方差不齐则采用Kruskal-Wallis H检验.组内不同时间点比较采用配对t检验.结果 实验第7和第14天,高渗液组BUT分别为(7.6±2.5)和(7.0±2.3)s,较第0天的(10.3±2.5)s显著缩短(t=5.800,4.950;均P＜0.01),与空白对照和生理盐水组相比亦明显缩短(F=8.030,P＜0.01).高渗液组结膜杯状细胞密度明显减少,第7和第14天分别为(19.5±16.6)和(32.3±18.2)个/mm2,与第0天(75.7±43.4)个/mm2相比,差异有统计学意义(t=5.319,2.970;均P＜0.05).高渗盐溶液滴眼第14天,光镜下可见结膜上皮下炎性细胞浸润,角膜上皮部分脱落;扫描电镜下可见角膜上皮细胞肿胀、脱屑、微绒毛减少;透射电镜下可见角膜上皮细胞问连接松弛,基底层上皮细胞内自噬空泡增多,结膜上皮微绒毛减少,杯状细胞内分泌颗粒减少;TUNEL检测结膜上皮可见明显的阳性染色;结膜组织免疫组织化学染色和免疫印迹检测则发现MUC5AC表达明显低于其他两组.生理盐水组与空白对照组的结膜杯状细胞密度及角结膜上皮细胞结构在实验中则均无明显变化.结论 眼表高渗压力可破坏角结膜上皮细胞结构,造成杯状细胞密度降低和MUC5AC表达水平下降,并导致泪膜稳定性下降.

Abstract：

Objective To investigate the effects of hyperosmotic stress on rabbit ocular surface and mucin 5AC (MUC5AC) expression. Methods Experimental study. Eighteen New Zealand white rabbits were randomly divided into three groups with equal number as hyperosmolar saline solution (HOSS,500 mmol/L) group, normal saline (NS, 308 mmol/L) group and blank control group respectively. In HOSS and NS groups, the HOSS and NS eye drops were instilled on bilateral eyes six times every day for 14 days. On day 0, 7 and 14, Schirmer Ⅰ test and tear break-up time (BUT) were measured and conjunctival impression cytology specimens were collected. On day 7 and 14, cornea and conjunctiva were harvested for Hematoxylin and Eosin (HE) staining, scanning and transmission electron microscopy observation and conjunctival TUNEL examination. On day 14, the conjunctiva were also harvested for immunology histological staining and western blot to evaluate the expression of MUC5AC. Results In HOSS group, the BUT on day 7 and 14 was (7.6±2. 5) and (7.0±2. 3) s respectively which was significantly shorter than the (10.3±2. 5) s on day 0(t=5. 800,4. 950; P ＜ 0.01), and also significantly shorter than the BUT in NS and control groups (F=8.030, P ＜ 0.01). But the Schirmer Ⅰ test value did not change obviously in and between all those three groups. The mean conjunctival goblet cell (GC) density in HOSS group on day 7 and 14 was (19.5±16.6) and (32.3±18.2) cells/mm2 respectively which was also significantly lower than the (75.7±43.4) cells/mm2 on day 0 (t=5.319,2. 970; P ＜ 0.05). However the GC density did not change obviously in other two groups with time. After instillation of HOSS for 14 days,subepithelial inflammatory cell infiltration was showed on conjunctival tissue specimens and decreased epithelial layers and evident desquamation were found in the cornea specimens by the HE staining. Under the electron microscope, decreased microvilli and loosened intercellular junction in the superficial epithelium and increased autophagic vesicles in basal epithelium were observed in the cornea in HOSS group; and decreased microvilli and mucous granules were found in the conjunctiva in HOSS group. Obvious TUNEL positive staining was showed in the conjunctiva in the HOSS group. Also the MUC5AC immunology histological staining and western blot indicated decreased MUC5AC protein expression in HOSS group. Conclusion Hyperosmotic stress destroyed the structure of ocular surface epithelium, induced the decrease of conjunctival goblet cell density and MUC5AC expression, and led to the decreased tear film stability.

The effects of hyperosmotic stress on rabbit ocular surface and mucin 5AC expression

Objective To investigate the effects of hyperosmotic stress on rabbit ocular surface and mucin 5AC (MUC5AC) expression. Methods Experimental study. Eighteen New Zealand white rabbits were randomly divided into three groups with equal number as hyperosmolar saline solution (HOSS,500 mmol/L) group, normal saline (NS, 308 mmol/L) group and blank control group respectively. In HOSS and NS groups, the HOSS and NS eye drops were instilled on bilateral eyes six times every day for 14 days. On day 0, 7 and 14, Schirmer Ⅰ test and tear break-up time (BUT) were measured and conjunctival impression cytology specimens were collected. On day 7 and 14, cornea and conjunctiva were harvested for Hematoxylin and Eosin (HE) staining, scanning and transmission electron microscopy observation and conjunctival TUNEL examination. On day 14, the conjunctiva were also harvested for immunology histological staining and western blot to evaluate the expression of MUC5AC. Results In HOSS group, the BUT on day 7 and 14 was (7.6±2. 5) and (7.0±2. 3) s respectively which was significantly shorter than the (10.3±2. 5) s on day 0(t=5. 800,4. 950; P ＜ 0.01), and also significantly shorter than the BUT in NS and control groups (F=8.030, P ＜ 0.01). But the Schirmer Ⅰ test value did not change obviously in and between all those three groups. The mean conjunctival goblet cell (GC) density in HOSS group on day 7 and 14 was (19.5±16.6) and (32.3±18.2) cells/mm2 respectively which was also significantly lower than the (75.7±43.4) cells/mm2 on day 0 (t=5.319,2. 970; P ＜ 0.05). However the GC density did not change obviously in other two groups with time. After instillation of HOSS for 14 days,subepithelial inflammatory cell infiltration was showed on conjunctival tissue specimens and decreased epithelial layers and evident desquamation were found in the cornea specimens by the HE staining. Under the electron microscope, decreased microvilli and loosened intercellular junction in the superficial epithelium and increased autophagic vesicles in basal epithelium were observed in the cornea in HOSS group; and decreased microvilli and mucous granules were found in the conjunctiva in HOSS group. Obvious TUNEL positive staining was showed in the conjunctiva in the HOSS group. Also the MUC5AC immunology histological staining and western blot indicated decreased MUC5AC protein expression in HOSS group. Conclusion Hyperosmotic stress destroyed the structure of ocular surface epithelium, induced the decrease of conjunctival goblet cell density and MUC5AC expression, and led to the decreased tear film stability.