Lentiviral Delivery of Small Hairpin RNA Targeting Connective Tissue Growth Factor Blocks Profibrotic Signaling in Tenon's Capsule Fibroblasts
Lentiviral Delivery of Small Hairpin RNA Targeting Connective Tissue Growth Factor Blocks Profibrotic Signaling in Tenon's Capsule Fibroblasts
复制标题
慢病毒递送靶向结缔组织生长因子的小发夹 RNA 阻断 Tenon 囊成纤维细胞中的促纤维化信号传导
DOI：
10.1167/iovs.16-19480
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发表时间：
2016-10-01
影响因子：
4.4
通讯作者：
Yuan, Huiping
中科院分区：
文献类型：
--
作者：
Lei, Dawei;Dong, Changgui;Yuan, Huiping
目的。小梁切除术是一种降低青光眼患者眼压的手术方法，在这种手术中，过度的瘢痕会导致滤过泡的失效，从而对手术结果产生不利影响。Tenon囊成纤维细胞(TCF)的高增殖和细胞外基质(ECM)沉积参与了这一过程，但调节TCF功能的内源性因素在很大程度上仍然难以捉摸。本研究旨在阐明结缔组织生长因子(CTGF)在TCF表型和信号转导中的作用。方法：采用实时定量聚合酶链式反应和免疫荧光技术检测结缔组织生长因子在瘢痕形成和无瘢痕形成的Tenon囊中的表达。CTGC的敲除是通过慢病毒传递小发夹状RNA实现的。CCK8法检测细胞增殖，流式细胞仪检测细胞周期进程，功能分析法检测细胞黏附、迁移和侵袭能力。结果：瘢痕形成的Tenon囊及其分离的成纤维细胞CTGF的表达明显高于非纤维化的Tenon囊成纤维细胞。慢病毒介导的小发夹状RNA靶向CTGF可抑制TCFs的增殖、黏附、迁移和侵袭，同时下调p38、核因子-kappaB、基质金属蛋白酶-2、细胞周期蛋白D1和I型胶原的表达。此外，慢病毒靶向CTGF可减少TCFs释放纤维化相关细胞因子，并抑制TCF诱导的巨噬细胞趋化。使用慢病毒载体靶向CTGF可能是预防小梁切除术后过度瘢痕形成的一种有前途的方法。
PURPOSES. Trabeculectomy is a surgical procedure for lowering intraocular pressure in glaucoma patients, in which excessive scarring leading to failure of the filtering bleb adversely affects the surgical outcome. Heightened Tenon's capsule fibroblast (TCF) proliferation and extracellular matrix (ECM) deposition are implicated in this process but endogenous factors that regulate TCF functions remain largely elusive. This study sought to elucidate the role of connective tissue growth factor (CTGF) in the regulation of TCF phenotypes and signaling.METHODS. Expression of CTGF in scarring and nonscarring Tenon's capsules was measured by real-time PCR and immunofluorescence. Knockdown of CTGC was achieved by lentivirus delivery of small-hairpin RNA. Cell proliferation was measured by CCK8, cell cycle progression, and apoptosis by flow cytometry, adhesion, migration, and invasion of TCF by functional assays in vitro. Proteins and cytokines related to fibrosis were measured by Western blot and ELISA, respectively.RESULTS. Expression of CTGF was significantly upregulated in scarring Tenon's capsules and their isolated fibroblasts when compared with the nonfibrotic counterparts. Functionally, targeting CTGF with lentivirus-delivered small-hairpin RNA inhibited the proliferation, adhesion, migration, and invasion of TCFs, accompanied by downregulation of p38 and nuclear factor-kappa B as well as matrix metalloproteinase-2, cyclin D1, and collagen I. In addition, lentiviral targeting of CTGF reduced the release of fibrosis-related cytokines from TCFs and inhibited TCF-conditioned, medium-induced macrophage chemotaxis.CONCLUSIONS. Our study supports a crucial role of CTGF in the regulation of TCF proliferation and ECM deposition. Targeting CTGF using lentiviral vector may be a promising approach for preventing excessive scarring after trabeculectomy.