<p>Flow cytometry antibodies and detection reagents.</p>

Purpose
Previous work demonstrated that supraphysiological glucose remodels TGF-β1 and NF-κB signaling in human limbal stromal cells (LSCs) and congenital aniridia-derived LSCs (AN-LSCs). The present study investigated whether the same metabolic stress also alters apoptotic pathways in these cells.
Methods
Primary LSCs (n = 12) and AN-LSCs (n = 8) were cultured for 48 hours in DMEM containing either normal (17.5 mM) or high (70 mM) glucose. Apoptosis was quantified by Annexin V/propidium iodide (PI) flow cytometry (FC). Expression levels of apoptosis-related genes—including CASP3/7/8/9/10, BCL2, BID, BAX, CDKN1A (p21), CDKN1B (p27), TNFα, XIAP, and BIRC5 (Survivin)—were assessed by qPCR. Protein levels of these markers were analyzed by FC, and TNFα protein concentrations in culture supernatants were measured by ELISA.
Results
High glucose significantly reduced the proportion of apoptotic cells in both LSCs (p = 0.0170) and AN-LSCs (p = 0.0181). In both cell types, CASP8 (p = 0.0448; p = 0.0171) and CASP10 (p = 0.0001; p = 0.0007) mRNA levels decreased, while XIAP (p = 0.0375; p = 0.0442) and BIRC5 (p = 0.0196; p = 0.0003) were upregulated. AN-LSCs additionally showed reductions in CASP3 (p = 0.0138) and CDKN1A (p = 0.0331), and exhibited lower BAX levels than LSCs under high glucose (p = 0.0255). Protein analysis corroborated these findings in AN-LSCs: Caspase-3 (p = 0.0154) and Caspase-8 (p = 0.0257) decreased, while Bcl-2 (p = 0.0284) and Survivin (p = 0.0467) levels increased. XIAP protein levels rose in both LSCs (p = 0.0451) and AN-LSCs (p = 0.0134).
Conclusions
A 48-hour exposure to 70 mM glucose induces a marked anti-apoptotic shift in human limbal stromal cells, more pronounced in cells derived from congenital aniridia patients. Together with previous evidence on TGF-β1 and NF-κB regulation, these findings suggest that limbal cells may mount an early protective response to metabolic stress, which could be harnessed therapeutically to manage aniridia-associated keratopathy through coordinated survival and stress pathways.

研究背景
既往研究表明，超生理浓度葡萄糖会重塑人角膜缘基质细胞（LSCs）与先天性无虹膜来源角膜缘基质细胞（AN-LSCs）中的转化生长因子-β1（TGF-β1）与核因子κB（NF-κB）信号通路。本研究旨在探究该代谢应激是否同样会改变上述细胞的凋亡通路。

实验方法
本研究纳入12例原代人角膜缘基质细胞与8例原代先天性无虹膜来源角膜缘基质细胞，将其分别培养于含正常葡萄糖浓度（17.5 mM）或高葡萄糖浓度（70 mM）的DMEM培养基中，培养时长为48小时。采用膜联蛋白V/碘化丙啶（PI）流式细胞术（FC）定量检测细胞凋亡水平；通过实时定量聚合酶链反应（qPCR）检测凋亡相关基因的表达，涉及基因包括CASP3/7/8/9/10、BCL2、BID、BAX、CDKN1A（p21）、CDKN1B（p27）、TNFα、XIAP及BIRC5（Survivin）；采用流式细胞术分析上述标志物的蛋白表达水平，并通过酶联免疫吸附实验（ELISA）测定细胞培养上清液中的TNFα蛋白浓度。

实验结果
高葡萄糖浓度可显著降低两种细胞的凋亡细胞占比：人角膜缘基质细胞组p=0.0170，先天性无虹膜来源角膜缘基质细胞组p=0.0181。在两种细胞中，CASP8（人角膜缘基质细胞组p=0.0448，先天性无虹膜来源组p=0.0171）与CASP10（人角膜缘基质细胞组p=0.0001，先天性无虹膜来源组p=0.0007）的mRNA水平均显著下调，而XIAP（人角膜缘基质细胞组p=0.0375，先天性无虹膜来源组p=0.0442）与BIRC5（人角膜缘基质细胞组p=0.0196，先天性无虹膜来源组p=0.0003）的mRNA水平则显著上调。
先天性无虹膜来源角膜缘基质细胞额外表现出CASP3（p=0.0138）与CDKN1A（p=0.0331）的mRNA水平降低，且在高葡萄糖培养条件下，其BAX蛋白水平低于人角膜缘基质细胞组（p=0.0255）。蛋白分析验证了先天性无虹膜来源组的上述结果：半胱氨酸天冬氨酸蛋白酶-3（Caspase-3，p=0.0154）与半胱氨酸天冬氨酸蛋白酶-8（Caspase-8，p=0.0257）的蛋白水平下降，而B细胞淋巴瘤因子2（Bcl-2，p=0.0284）与生存素（Survivin，p=0.0467）的蛋白水平上升。两种细胞的XIAP蛋白水平均显著升高：人角膜缘基质细胞组p=0.0451，先天性无虹膜来源组p=0.0134。

研究结论
经70 mM葡萄糖处理48小时后，人角膜缘基质细胞会出现显著的抗凋亡表型转变，且该效应在先天性无虹膜患者来源的细胞中更为明显。结合既往关于TGF-β1与NF-κB调控的研究证据，本研究结果提示，角膜缘细胞可能对代谢应激产生早期保护性应答，该应答可通过协同调控生存与应激通路，为治疗无虹膜相关性角膜病提供潜在的治疗策略。