低剂量微丝解聚剂对地塞米松诱导的牛小梁细胞骨架及基质蛋白TSP-1的影响

Effects of low dose of latrunculin-A on actin cytoskeleton and extracellular matrix protein TSP-1 in dexamethasone-induced bovine TMCs

目的研究低剂量的肌动蛋白微丝解聚剂Latrunculin(LAT)-A对地塞米松诱导的牛小梁细胞肌动蛋白细胞骨架及细胞外基质蛋白TSP-1产生的影响。方法体外培养牛眼小梁细胞,分别用地塞米松和LAT-A在不同条件下孵育,免疫荧光显微镜观察细胞骨架形态和结构的改变并用免疫组织化学法对TSP-1的表达进行定位和半定量分析。结果培养的小梁细胞NSE染色阳性,ⅧR∶Ag染色阴性,TSP-1染色阳性。经地塞米松诱导后微丝重组杂乱,细胞外TSP-1表达减弱。LAT-A可部分逆转此重组,促进其恢复;LAT-A可抑制TSP-1表达,且随作用时间的延长抑制作用更强。结论低剂量的LAT-A可以逆转由地塞米松诱导的细胞骨架的改变,并对TSP-1的产生有抑制作用。

Objective Glaucoma is the leading cause of irreversible blindness. Now there is no specifically effective treatment against it in clinic. We determined the effects of a low dose of the actin-disrupting agent latrunculin （ LAT）-A on dexamethasone （DEX） -induced changes in actin organization and extracellular matrix proteins in cultured bovine trabecular meshwork cells（BTMCs）. Methods BTMCs were primarily cultured with tissue method and subcuhured to the 3rd passage. The 3rd generation of cells were divided into untreated group,DEX treated for 2 weeks group,DEX for 2 weeks ＋ LAT-A for 2 hours group and DEX for 1 week ＋ LAT-A for 1 week group. The marking of F-actin in the cell was performed by FITC-phallodin and observed under the Olympus fluorescence microscope. The expression of LAT-A was detected by immunohistochemistry. Results The primarily cultured BTMCs showed variable appearance, but the 3rd passage of BTMCs had stable shape with framework and affluent cytoplasm,and these cells inosculated as confluent monolayer like flat epithelium. The 3rd passage of cells were identified by immunohistochemistry. The cells treated with DEX showed actin cytoskeletal reorganization and formation of cross-linked actin networks. In DEX treated cells,actin fiber bundles were highly concentrated at the periphery of the cell with few actin filaments in the central area. After LAT-A treatment,there was mild disorganization of actin cytoskeleton,and actin staining in DMSO treated cells was comparable to untreated cells. Conclusion This study suggests that LAT-A at low dose can avoid the ocular high pressure induced by DEX. It may be useful in the treatment and prevention of steroid and other glaucoma.