Wang Qun, Wei Chao, Ma Li, Wang Xin, Li Lin, Zhou Qingjun, Shi Weiyun
Medical College, Qingdao University, 38 Dengzhou Road, Qingdao, 266021, China.
State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong Academy of Medical Sciences, Yanerdao Road, Qingdao, 266071, China.
Graefes Arch Clin Exp Ophthalmol. 2018 Apr;256(4):709-715. doi: 10.1007/s00417-018-3913-0. Epub 2018 Feb 26.
Endothelial dysfunction accounts for 50% of total corneal transplantation failures, suggesting that corneal endothelial damage is the leading cause of graft failure. Tumor necrosis factor-α (TNF-α) is known to contribute to the negative regulation of corneal transplantation, but how it does so remains unclear. Here, we report a regulatory loop involving TNF-α, TNF-α-induced protein 8 like 2 (TNFAIP8L2 or TIPE2), and apoptosis during corneal graft rejection.
We established mice models of penetrating keratoplasty to verify whether the quantification of TNF-α in allogeneic corneas is enhanced through ELISA assay and immunofluorescence staining. In cornea tissues, we obtained corneal endothelium and measured apoptosis of the removed cells. Meanwhile, quantitative real-time PCR and Western blotting were used to detect the mRNA and protein expression of TIPE2. In human corneal endothelial cells, we verified the conclusions through some experiments. By specifically knocking down TIPE2, we detected the importance of TIPE2 in TNF-α-triggered apoptosis.
In mice models, TNF-α was higher in the cornea and aqueous humor in allograft group and TNF-α elevation increased the apoptosis of the corneal endothelium. In addition, high levels of TIPE2 were found in allograft rejection models following TNF-α elevation. In human corneal endothelial cells (HCECs), TNF-α clearly augments TIPE2 expression and promotes cell apoptosis through upregulating TIPE2 transcription. Knocking down markedly decreased cell apoptosis.
Our study identifies the molecular mechanisms underlying the interplay of TNF-α, TIPE2, and apoptosis during allograft rejection, and it suggests that both TNF-α and TIPE2 might be potential targets for the successfully grafted corneal endothelium.
内皮功能障碍占角膜移植失败总数的50%，这表明角膜内皮损伤是移植失败的主要原因。已知肿瘤坏死因子-α（TNF-α）会对角膜移植产生负调控作用，但其具体机制尚不清楚。在此，我们报告了一个在角膜移植排斥反应过程中涉及TNF-α、TNF-α诱导蛋白8样2（TNFAIP8L2或TIPE2）和细胞凋亡的调控环路。
我们建立了穿透性角膜移植小鼠模型，通过酶联免疫吸附测定（ELISA）和免疫荧光染色来验证同种异体角膜中TNF-α的定量是否增加。在角膜组织中，我们获取角膜内皮并检测去除细胞的凋亡情况。同时，使用定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测TIPE2的mRNA和蛋白表达。在人角膜内皮细胞中，我们通过一些实验验证了这些结论。通过特异性敲低TIPE2，我们检测了TIPE2在TNF-α触发的细胞凋亡中的重要性。
在小鼠模型中，同种异体移植组角膜和房水中的TNF-α水平较高，TNF-α升高增加了角膜内皮细胞的凋亡。此外，在TNF-α升高后的同种异体移植排斥模型中发现了高水平的TIPE2。在人角膜内皮细胞（HCECs）中，TNF-α明显增强TIPE2表达，并通过上调TIPE2转录促进细胞凋亡。敲低TIPE2可显著减少细胞凋亡。
我们的研究确定了同种异体移植排斥反应过程中TNF-α、TIPE2和细胞凋亡相互作用的分子机制，并表明TNF-α和TIPE2可能都是成功移植角膜内皮的潜在靶点。
