甲状腺相关眼病患者眼外肌中胶原蛋白和HSP47的表达

Expression of collagen and heat shock protein 47 in extraocular muscle tissues of patients with thyroid-associated ophthalmopathy

目的 探讨甲状腺相关眼病(TAO)患者眼外肌中胶原蛋白(Ⅰ、Ⅲ、Ⅴ型)和热休克蛋白47(HSP47)的表达特点。方法 收集2019年5月至2019年9月在中山大学眼科中心明确诊断为静止期TAO患者4例右眼眼外肌(TAO组),同期收集于我院行斜视矫正手术的共同性斜视患者4例右眼眼外肌(对照组)。对两组眼外肌分别进行HE染色、Masson染色分析样本中胶原蛋白(Ⅰ、Ⅲ、Ⅴ型)相对表达和纤维化的程度。实时荧光定量聚合酶链反应(qRT-PCR)检测两组眼外肌中胶原蛋白(Ⅰ、Ⅲ、Ⅴ型)和HSP47的mRNA相对表达量;Western blot检测两组眼外肌中胶原蛋白(Ⅰ、Ⅲ、Ⅴ型)和HSP47表达情况。结果 Masson染色结果显示,TAO组和对照组眼外肌中均显示大量胶原纤维,但对照组的眼外肌的形态模糊,胶原纤维分布较散乱;而TAO组眼外肌形态较清晰,胶原纤维排列较密集。HE染色结果显示,对照组眼外肌形态不清,肌纤维少且排列紊乱;TAO组眼外肌形态较清晰,肌纤维分布较集中,排列比较规则,横截面积大小不一。Western blot检测结果显示,TAO组和对照组眼外肌中胶原蛋白(Ⅰ、Ⅲ、Ⅴ型)和HSP47均有表达,但对照组眼外肌中胶原蛋白(Ⅰ、Ⅲ、Ⅴ型)相对表达量均高于TAO组(均为P<0.05)。Pearson相关分析结果显示,TAO组与对照组眼外肌中HSP47分别与两组眼外肌中的胶原蛋白(Ⅰ、Ⅲ、Ⅴ型)表达均呈正相关(均为P<0.01)。qRT-PCR检测结果显示,TAO组与对照组眼外肌中胶原蛋白(Ⅰ、Ⅲ、Ⅴ型)和HSP47的mRNA相对表达量差异均无统计学意义(均为P>0.05)。结论 HSP47可在TAO患者眼外肌中表达且眼外肌的纤维化可能与HSP47蛋白表达有关。

Objective To discover the expression characteristics of collagen(typesⅠ,ⅢandⅤ)and heat shock protein 47(HSP47)in extraocular muscle tissues of patients with thyroid-associated ophthalmopathy(TAO).Methods Extraocular muscle tissues of four right eyes were collected,respectively,from patients with resting TAO diagnosed at the Eye Center of Sun Yat-sen University between May 2019 and September 2019(TAO group)and from patients who underwent strabismus correction surgery at our hospital for concomitant strabismus during the same period(control group).The hematoxylin-eosin(HE)staining and Masson staining were performed on the extraocular muscle tissues in the two groups to assess the collagen(types I,Ⅲand V)expression and fibrosis.The relative mRNA expression levels of collagen(types I,Ⅲand V)and HSP47 in the extraocular muscle tissues were measured by quantitative real-time polymerase chain reaction(qRT-PCR).The expression levels of collagen(typesⅠ,ⅢandⅤ)and HSP47 in the extraocular muscle tissues were measured by Western blot.Results Masson staining revealed many collagen fibers in the extraocular muscle tissues in both the TAO and control groups.The morphology of extraocular muscle tissues in the control group was vague,and collagen fibers were distributed diffusely;however,in the TAO group,the morphology of extraocular muscle tissues was clear,and collagen fibers were distributed densely.HE staining showed that the extraocular muscle tissues in the control group were unclear in morphology,and muscle fibers were few and irregularly distributed;however,in the TAO group,the extraocular muscle tissues were clear in morphology,and muscle fibers varied in the cross-sectional area but were distributed densely and regularly.Western blot showed that collagen(typesⅠ,ⅢandⅤ)and HSP47 were expressed in the extraocular muscle tissues in both the TAO and control groups,but the expression levels of collagen(typesⅠ,ⅢandⅤ)in the control group were higher than those in the TAO group(all P<0.05).Pearson analysis showed that the expression of HSP47 was positively correlated with that of collagen(typesⅠ,ⅢandⅤ)in both the TAO and control groups,respectively(all P<0.01).The qRT-PCR data showed that there was no statistically significant difference in the relative mRNA expression levels of collagen(typesⅠ,ⅢandⅤ)and HSP47 between the two groups(all P>0.05).Conclusion HSP47 is expressed in the extraocular muscle tissues of TAO patients.The fibrosis of the extraocular muscle tissues may be related to the HSP47 protein expression.