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Abstract：
Collagen crosslinking (CXL) is a widely used treatment to halt the progression of keratoconus (KC). Unfortunately, a significant number of patients with progressive KC will not qualify for CXL, including those with corneas thinner than 400 µm. The present study aimed to investigate the molecular effects of CXL using in vitro models, mirroring the normal, as well as thinner corneal stroma seen in KCs. Primary human corneal stromal cells were isolated from healthy (HCFs) and keratoconus (HKCs) donors. Cells were cultured and stimulated with stable Vitamin C resulting in 3D self-assembled extracellular matrix (ECM), cell-embedded, constructs. CXL was performed on (a) thin ECM with CXL performed at week 2 and (b) normal ECM with CXL performed at week 4. Constructs without CXL served as controls. All constructs were processed for protein analysis. The results showed modulation of Wnt signaling, following CXL treatment, as measured by the protein levels of Wnt7b and Wnt10a, correlated to the expression of α-smooth muscle actin (SMA). Further, the expression of a recently identified KC biomarker candidate, prolactin-induced protein (PIP), was positively impacted by CXL in HKCs. CXL-driven upregulation of PGC-1 and the downregulation of SRC and Cyclin D1 in HKCs were also noted. Although the cellular/molecular impacts of CXL are largely understudied, our studies provide an approximation to the complex mechanisms of KC and CXL. Further studies are warranted to determine factors influencing CXL outcomes.
摘要：
胶原蛋白交联（CXL）是一种广泛用于阻止圆锥角膜（KC）进展的治疗方法。不幸的是,大量进行性KC患者不符合CXL，包括角膜厚度小于400微米的.本研究旨在利用体外模型研究CXL的分子效应,镜像正常，以及在KCs中看到的较薄的角膜基质。从健康(HCFs)和圆锥角膜(HKCs)供体分离原代人角膜基质细胞。培养细胞并用稳定的维生素C刺激，产生3D自组装的细胞外基质（ECM），细胞嵌入，构造。对(a)在第2周进行CXL的细ECM和(b)在第4周进行CXL的正常ECM进行CXL。没有CXL的构建体用作对照。处理所有构建体用于蛋白质分析。结果显示Wnt信号的调节，CXL治疗后，通过Wnt7b和Wnt10a的蛋白质水平测量，与α-平滑肌肌动蛋白（SMA）的表达相关。Further,最近确定的KC生物标志物候选的表达，催乳素诱导蛋白（PIP），在HKCS中受到CXL的积极影响。还注意到CXL驱动的PGC-1上调以及HKCs中SRC和CyclinD1的下调。尽管CXL的细胞/分子影响在很大程度上被研究不足，我们的研究为KC和CXL的复杂机制提供了近似。需要进一步的研究来确定影响CXL结局的因素。
