从内质网应激途径探讨羟喜树碱诱导人Tenon's囊成纤维细胞凋亡的机制

Scarring in surgical area is the main reason for the failure of trabeculectomy and other anti-glaucoma surgeries. Recent studies shown that hydroxycamptothecin (HCPT) had a significant effect on preventing human Tenon's capsule fibroblasts proliferation. Furthermore, experiments demonstrated that human Tenon's capsule fibroblasts apoptosis were induced by cell receptor pathway and mitochondrial pathway. However, after blocking the above two pathways, there was still apoptosis in the Tenon's capsule fibroblasts ， which suggested that there miaght be other apoptosis pathways involved in this activity. This experiment is designed to investigate whether the endoplasmic reticulum stress pathway is involved in this apoptotic event. On one hand, we will examine key molecules' expression change in the endoplasmic reticulum stress pathway, including CHOP , caspase-12 and so on. On the other hand, we will test the apoptosis change of human Tenon's capsule fibroblasts after inhibating the expression of CHOP and caspase-12. Thus, we would like to identify whether endoplasmic reticulum stress pathway is involved in the human Tenon's capsule fibroblasts apoptosis. And It is clarified that the mechanism of HCPT on preventing fibroblasts proliferation, in order to supply a new method for the improvement of glaucoma surgeries and increase the success rate of surgeries.

手术区瘢痕化，无法形成有效滤过泡，是小梁切除术等抗青光眼手术失败的主要原因。近年来研究发现羟喜树碱（HCPT）预防人Tenon's囊成纤维细胞增殖有显著效果，且实验证明其通过细胞受体途径和线粒体途径诱导人Tenon's囊成纤维细胞凋亡，但进一步实验发现，阻断上述两种途径后仍有Tenon's囊成纤维细胞发生凋亡，提示有其他凋亡途径参与其中。故提出假说，内质网应激途径参与诱导人Tenon's囊成纤维细胞凋亡。通过观察HCPT作用后人Tenon's囊成纤维细胞中CHOP,caspase-12等内质网应激的关键分子的表达水平的变化，以及抑制CHOP，caspase-12表达后观察成纤维细胞凋亡水平的变化，来分析HCPT是否通过内质网应激途径引起成纤维细胞凋亡的发生，进而阐明HCPT预防人Tenon's囊成纤维细胞增殖的具体机制。为临床改良抗青光眼手术，提高手术成功率提供一种新方法。

滤过道瘢痕化，人Tenon’s囊成纤维细胞 ( HTFs ) 增殖是青光眼滤过手术失败的主要原因。近年来研究发现羟喜树碱（HCPT）预防Tenon’s囊成纤维细胞增殖有显著效果，且实验证明其通过细胞受体途径和线粒体途径诱导人Tenon’s囊成纤维细胞凋亡，但进一步实验发现，阻断上述两种途径和线粒体途径诱导人Tenon’s囊成纤维细胞凋亡，提示有其他凋亡途径参与其中。已有研究证实在某些因素比如抗代谢药物的刺激下，细胞的自噬和凋亡现象可以同时被观察到。因此我们提出假设，内质网应激途径参与诱导人Tenon’s囊成纤维细胞凋亡，且羟喜树碱在诱导HTFs凋亡的同时，对另一种程序性细胞死亡－自噬也产生一定影响。通过观察HCPT作用后人Tenon’s囊成纤维细胞中CHOP,caspase-12等内质网应激的关键分子，以及抑制CHOP,caspase-12表达后观察成纤维细胞凋亡水平的变化，证实HCPT通过内质网应激途径引起成纤维细胞凋亡的发生。通过检测自噬相关蛋白Beclin 1 、LC3等的表达水平变化，mRFP-GFP-LC3 腺病毒载体转染评价自噬水平，证实了羟喜树碱能诱导人Tenon’s囊成纤维细胞发生显著自噬。我们发现miR-216b通过与Beclin 1结合及相互作用对羟喜树碱诱导的HTFs的自噬和凋亡起调节作用，进而阐明HCPT预防人Tenon’s囊成纤维细胞增殖的具体机制。为临床改良青光眼手术，提高手术成功率提供一种新方法。