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Abstract：
Advanced glycation end products (AGEs) form extracellular crosslinking with collagenous proteins, which contributes to the development of diabetic complications. In this study, AGEs-related pentosidine (PENT) crosslinks-induced structural and biochemical changes are studied using multimodal multiphoton imaging, Raman spectroscopy and atomic force microscopy (AFM). Decellularized equine pericardium (EP) was glycated with four ribose concentrations ranging between 5 and 200 mM and monitored for up to 30 days. Two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) microscopic imaging probed elastin and collagen fibers, respectively. The glycated EP showed a decrease in the SHG intensities associated with loss of non-centrosymmetry of collagen and an increase of TPEF intensities associated with PENT crosslinks upon glycation. TPEF signals from elastin fibers were unaffected. A three-dimensional reconstruction with SHG + TPEF z-stack images visualized the distribution of collagen and elastin within the EP volume matrix. In addition, Raman spectroscopy (RS) detected changes in collagen-related bands and discriminated glycated from untreated EP. Furthermore, AFM scans showed that the roughness increases and the D-unit structure of fibers remained unchanged during glycation. The PENT crosslinked-induced changes are discussed in the context of previous studies of glutaraldehyde- and genipin-induced crosslinking and collagenase-induced digestion of collagen. We conclude that TPEF, SHG, RS, and AFM are effective, label-free, and non-destructive methods to investigate glycated tissues, differentiate crosslinking processes, and characterize general collagen-associated and disease-related changes, in particular by their RS fingerprints.
摘要：
晚期糖基化终产物（AGEs）与胶原蛋白形成细胞外交联，这有助于糖尿病并发症的发展。在这项研究中,使用多模态多光子成像研究了与AGEs相关的戊糖苷（PENT）交联引起的结构和生化变化，拉曼光谱和原子力显微镜(AFM)。将脱细胞马心包(EP)用4种浓度在5至200mM之间的核糖糖化,并监测长达30天。双光子激发荧光（TPEF）和二次谐波发生（SHG）显微成像探测弹性蛋白和胶原纤维，分别。糖化EP显示与胶原的非中心对称性的丧失相关的SHG强度的降低和与糖化后的PENT交联相关的TPEF强度的增加。来自弹性蛋白纤维的TPEF信号不受影响。使用SHGTPEFz堆叠图像的三维重建可视化了EP体积基质中胶原蛋白和弹性蛋白的分布。此外,拉曼光谱（RS）检测到胶原蛋白相关条带的变化，并将糖化与未处理的EP区分开。此外，AFM扫描显示，糖化过程中粗糙度增加，纤维的D单元结构保持不变。在戊二醛和京尼平诱导的交联以及胶原酶诱导的胶原蛋白消化的先前研究的背景下，讨论了PENT交联诱导的变化。我们得出结论，TPEF，SHG,RS,AFM是有效的，无标签，和非破坏性的方法来研究糖化组织，区分交联过程，并表征一般胶原蛋白相关和疾病相关的变化，特别是他们的RS指纹。
