白细胞介素-1α对猪小梁细胞基质金属蛋白酶／基质金属蛋白酶抑制剂表达的影响

Effects of interleukin-1α on the expression of matrix metalloproteinases and tissue matrix metaiioproteinase inhibitors in swine trabecular meshwork cells

背景房水外流通路的阻塞或小梁网细胞外基质（ECM）的异常堆积导致房水流畅系数降低是引起眼压升高的原因之一，基质金属蛋白酶（MMPs）和组织金属蛋白酶抑制剂（TIMPs）的平衡对ECM的代谢至关重要，白细胞介素-1α（IL-1α）可以通过调节MMPs的表达而调节房水的外流。目的研究猪重组IL-1α对体外培养的猪眼小梁细胞MMP-2、MMP-3及TIMP-1表达的影响。方法从猪眼取出带有小梁组织的巩膜，用组织块培养法培养小梁细胞并进行传代，第3代的猪小梁细胞应用纤维连接蛋白（FN）和层黏连蛋白（LM）进行鉴定。第3代小梁细胞血清饥饿培养24h后分为2组，对照组加入无血清培养基，IL组加入10m／L IL-1α，分别培养30min。采用细胞免疫化学法分析IL组MMP-2、MMP-3及TIMP-1蛋白在培养小梁细胞中的表达；采用逆转录聚合酶链反应（RT—PCR）法检测小梁细胞中MMP-2mRNA、MMP-3mRNA及TIMP-1 mRNA的表达，并与对照组的检测结果进行比较。结果传3代的细胞对FN和LM呈现阳性反应。与对照组比较，IL组小梁细胞中MMP-3和TIMP-1蛋白的表达量（A值）明显升高，差异均有统计学意义（t=-7．694、t=-5．199，P〈0．05），但2组间小梁细胞中MMP-2表达的差异无统计学意义（t=-2．365，P〉0．05）。IL组小梁细胞中MMP-3mRNA和TIMP-1mRNA的表达量（A值）明显高于对照组，差异均有统计学意义（t=-3．025、t=-1．921，P〈0．05），而2组间小梁细胞中MMP-2mRNA的表达差异无统计学意义（t=-1．173，P〉0．05）。结论外源性IL-1α能增加猪眼小梁细胞中MMP-3、TIMP-1的表达，引起MMP-3／TIMP-1平衡改变，促进小梁网ECM的分解，增加房水外流，而对MMP-2的表达无影响。

Background Obstruction of aqueous humor out flow pathway or abnormality of the extracellular matrix（ECM） of trabecular meshwork cells causes high intraocular pressure. The balance of matrix metalloproteinases （MMPs） and tissue inhibitors of matrix metalloproteinases（TIMPs） is critical for the metabolism of ECM. Interleukin-1α（IL-1α） can influence outflow of aqueous humor by regulating MMPs level. Objective This study was to investigate the effect of interleukin-1α on the expression of MMP-2,MMP-3 and TIMP-1 in cultured swine trabecular meshwork cells. Methods Swine sclera with trabecular meshwork tissue was isolated from 20 swine eyes and cultured with explant cultured method. Cultured cells were passaged and third generation cells were identified by fibronectin （FN） and laminin （LN） staining. After 24 hours of serum starvation, trabecular meshwork cells treated with IL-1α at the concentration of 10 mg/L were regarded as the IL group, and serum-free culture medium used to treat trabecular meshwork cells was regarded as the control group. The expression of MMP-2,MMP-3 and TIMP-1 proteins in trabecular meshwork cells were detected by immunohistochemistry, and the expression of MMP-2 mRNA, MMP-3 mRNA and TIMP-1 mRNA were detected by RT-PCR. The examination results were compared between the two groups. Results The third generation of cells were positive for FN and LM. Compared with the control group,the expression levels of MMP-3 and TIMP-1 proteins（A value） in trabecular meshwork cells were significantly higher in the IL group than the control group （ t = -7. 694, t = -5. 199, P〈0.05 ） , but no obvious difference was found in the expression of MMP-2 between the two groups（ t=-2. 365 ,P〉0. 05 ）. The higher expression levels in MMP-3 mRNA and TIMP-1 mRNA （A value） in trabecular meshwork cells were seen in comparison with the control group （t = -3. 025 ,t=-1 921 ,P〈0.05）. However, similar results were found in the expression of MMP-2 mRNA between the two groups（ t = - 1. 173, P〉 0.05 ）. Conclusions The overexpression of MMP-3 and TIMP-1 proteins and their mRNA leads to the imbalance of MMP-3/TIMP-1 and promotes the decomposition of ECM in the trabeeular meshwork, and therefore increases aqueous outflow.