热休克蛋白47在大鼠抗青光眼滤过术后滤过泡中的动态表达

Dynamic expression of heat shock protein 47 in filtering bleb following the glaucomatous filtering surgery in rat

背景抗青光眼滤过术后抗滤过泡的瘢痕化是眼科研究的热点和难点，研究已证实热休克蛋白47（HSP47）的表达上调与人体多个器官和组织胶原纤维的合成以及组织纤维化过程密切相关，但其与青光眼术后滤过泡的纤维化是否有关尚不清楚。目的探讨HSP47与大鼠抗青光眼滤过术后滤过泡组织纤维增生的关系。方法健康雄性SPF级Spargue—Dawley（SD）大鼠10只，所有大鼠右眼均行常规抗青光眼滤过手术，左眼为正常对照眼。应用随机数字法将术眼随机分为术后7d组和14d组，每组5只。术后每日在裂隙灯显微镜下观察滤过泡形态及眼前节情况。分别于术后7d和14d采用过量麻醉法处死大鼠，并制备正常对照眼结膜组织和手术眼术区组织标本，分别行苏木精-伊红染色和免疫组织化学染色，检测组织标本中胶原纤维的增生及HSP47在滤过泡中的表达情况，计算标本中HSP47阳性细胞数占总细胞数的百分比，即阳性细胞百分比。结果10只大鼠右眼术后均可见弥散隆起的结膜滤过泡，局部炎症反应轻微。苏木精一伊红染色结果表明，与正常对照眼结膜下组织比较，抗青光跟滤过术后术跟滤过泡组织中可见成纤维细胞增生，胶原纤维增多，呈疏松束状至致密束状排列。HSP47在SD大鼠正常对照眼结膜组织的成纤维细胞和血管内皮细胞的细胞质中可见棕黄色染色，而在滤过术后7d的滤过泡组织中，HSP47在阳性细胞中染色强度较正常对照组增强，HSP47阳性细胞百分比为（56．40±1．12）％，与正常对照组的（13．70±0．74）％相比，差异有统计学意义（P=0．000）；滤过术后14d时HSP47在滤过泡中的阳性细胞百分比为（23．90±0．76）％，与术后7d时比较，差异有统计学意义（P=0．000），但是仍高于正常对照组，差异有统计学意义（P=0．000）。结论SD大鼠抗青光眼滤过术后HSP47的表达量与滤过泡中成纤维细胞的活性相一致，提示HSP47在胶原合成过程中发挥作用。

Background Anti-scarring is a focus following glaucoma filtering surgery. It has been verified that expression of heat shock protein 47 （HSP47） is always correlated with that of various types of collagens and closely related with the collagen-related diseases including fibrosis in various organs. However, the relationship between the expression of HSP47 and fibrosis of filtering bleb is unclear. Objective Present study was to investigate the dynamic expression of HSP47 in subconjunctival filtering bleb after anti-glaucomatous filtering surgery. Methods Filtering surgery was performed on the right eyes of 10 SPF male Spargue-Dawley （SD） rats. Then the operative eyes were randomized into the postoperative 7-day group and 14-day group according to the randomized number table,and the left eyes of the rats served as the normal control group. The shape of filtering blebs and the ocular anterior segment response were examined under microscope daily. The animals were sacrificed by excessive anesthesia on day 7 and 14 after operation, respectively, and the specimens were prepared of the eyes. Regular histopathological examination was performed to observe the collagen fibrosis change, and immunobistochemistry was carried out to assay the dynamic expression of HSP47 depended on the fibrosis. Percentage of positive cells for HSP47 were calculated. The use and care of the experimental animals complied with the Regulation for the Administration of Affair Concerning the Experimental Animals by State Science and Technology Committee. Results Diffuse subconjunctival blebs were seen postoperatively in 10 eyes after surgery with mild inflammatory response in ocular anterior segment. Hematoxylin-eosin staining showed that compared with the specimens of the normal control group, fibroblasts obviously proliferated and collagenous fibers increased in the specimens of operative group. The HSP47 protein was less expressed in cytoplasm of many fibroblasts and less vascular endothelial cells in the normal rat specimens. The expressing intensity of HSP47 protein was enhanced. However,the expression intensity was weaker in the postoperative 14-day group in comparison with the 7-day group. The percentage of positive ceils for HSP47 in the filtering blebs 7 days after operation was （56.40±1.12） % ,that in the normal control group was （ 13.70±0. 74） % , with a significant difference between them （P = 0. 000）. In the postoperative 14-day group,the percentage of positive ceils for HSP47 reduced from （56.40＋1. 12）% to （23.90±0. 76）% （P=0. 000） ,but it was still higher than that of the normal control group （ P = 0. 000）. Conclusions Expression intensity of HSP47 alters as the activity of fibroblasts in filtering bleb, suggesting that HSP47 plays a role in collagen-fibrosis following filtering surgery in SD rats.