光交联的多糖AFG-GelMA水凝胶愈合糖尿病伤口的药效学研究

	光交联的多糖AFG-GelMA水凝胶愈合糖尿病伤口的药效学研究
	宋雪梅
导师	吴明一
关键词	糖尿病伤口愈合，蜗牛糖胺聚糖，水凝胶，慢性炎症，巨噬细胞 Diabetic wound healing, Snail glycosaminoglycan, Hydrogels, Chronic inflammation, Macrophages
摘要	正常伤口的愈合是一个高度有序的过程，涉及止血、炎症、增殖和重塑四个独立又相互重叠的阶段。研究发现，过度和持续的炎症导致伤口无法进入增殖和重塑阶段，组织再生困难，创伤无法愈合而形成慢性伤口。作为糖尿病三大并发症之一的糖尿病足溃疡，是临床上最常见的慢性伤口。糖尿病患者伤口常伴随着大量的炎性细胞浸润、促炎因子过量表达、活性氧自由基和蛋白酶含量过高等病理现象，使伤口长期处于炎症阶段导致其难以愈合。因此，调节伤口的炎症促进其向增殖阶段的转变，已成为糖尿病慢性伤口治疗的重要研究方向。本课题前期研究发现，从白玉蜗牛（Achatina fulica var.）中提取的一种新型蜗牛糖胺聚糖（AFG），能够显著减轻早期炎症反应，促进糖尿病大鼠皮肤伤口的愈合。为了提高给药的便利性，采用光交联的方法制备了一种基于AFG和甲基丙烯酰化明胶（GelMA）的AFG/G30水凝胶。该水凝胶制备简便，具有良好的组织粘附性和生物相容性。本文进一步运用链脲佐菌素诱导的糖尿病大鼠伤口模型，对AFG/G30水凝胶的治疗效果进行评价，并探讨其愈合糖尿病创面的作用机制。以生理盐水（Control）、G30和HA/G30水凝胶作为对照，考察AFG/G30水凝胶促进糖尿病大鼠创面愈合的药效。对术后第0、3、6、10、14天的愈合进度的定量分析表明，用AFG/G30水凝胶治疗的组优于其他组。此外，通过H&E、Masson组织染色和α-SMA/CD31免疫荧光染色，显示AFG/G30水凝胶能有效减轻伤口的炎症和水肿症状，刺激新血管形成、上皮再生和胶原蛋白的沉积。动物实验结果表明，AFG/G30水凝胶对糖尿病伤口具有良好的抗炎和促愈合的作用。利用生物信息学和分子生物学方法，对AFG/G30水凝胶愈合糖尿病伤口的作用机制进行初步探讨。选取术后第3天和第10天的创面组织进行转录组测序，鉴定差异表达基因及潜在的信号通路。一共筛选出1065个差异表达基因，其中第3天AFG/G30水凝胶组中差异表达基因的数量是另外两个水凝胶组的3倍，有590个上调基因和593个下调基因，且G30和HA/G30水凝胶组大部分的下调基因与AFG/G30组的下调基因相重叠；GO/KEGG显著性富集分析显示，术后第3天的下调基因主要与炎症、免疫反应和趋化因子介导的信号通路相关；选择富集通路中35个重要的差异表达基因进行聚类分析，发现第3天Control组中的炎症基因几乎都高表达，而AFG/G30水凝胶抑制了相关炎症基因的表达并使组织再生相关基因的表达水平明显提高。通过RT-qPCR法对相关基因进行验证，得到结果基本一致。此外，部分炎性细胞因子的检测结果也表明，AFG/G30水凝胶在第3天能够减少相关炎症因子（IL-6、TNF-α、IL-10和IL-1β）的分泌。因此，AFG/G30水凝胶在伤口愈合早期对炎症反应的调节作用，是促进糖尿病伤口愈合的关键。巨噬细胞的表型变化是伤口从炎症向增殖阶段转换的关键因素。利用流式细胞术、CD206/CD86免疫荧光双染色和RT-qPCR检测进一步探究AFG/G30水凝胶对伤口组织中巨噬细胞表型的影响，结果显示AFG/G30水凝胶能促进巨噬细胞向M2型巨噬细胞转化，且M2型巨噬细胞标志物CD206基因的表达水平也明显升高。同时，以RAW264.7巨噬细胞为研究对象展开体外实验，证明AFG/G30水凝胶能够抑制LPS诱导巨噬细胞向M1型极化，而促使巨噬细胞向M2型转化。推测AFG/G30水凝胶极有可能通过调节M2型巨噬细胞的极化以减轻糖尿病伤口部位的炎症反应。综上所述，AFG/G30水凝胶通过介导与炎症相关的生物学过程，以调节糖尿病伤口的微环境，有效促进血管新生、上皮再生和胶原蛋白沉积，最终加速糖尿病伤口的愈合。本文研究为AFG/G30水凝胶作为一种新颖的糖尿病慢性伤口愈合潜在候选药物的进一步开发奠定了理论基础。; Normal wound healing is an orchestrated process involving four dependent and overlapping stages of hemostasis, inflammation, proliferation and remodeling. The excessive and persistent inflammation results in failure to enter the proliferative and remodeling phases, which hinders tissue regeneration and wound healing, leading to chronic wounds. The diabetic foot ulcer, one of the three major complications of diabetes, is the most common chronic wound in clinical practice. Diabetic wounds are generally accompanied by massive inflammatory cell infiltration, overexpression of pro-inflammatory cytokines, and high levels of reactive oxygen radicals and proteases, which causes a long-term inflammatory phase to prevent wound closure. Therefore, regulating inflammation and promoting the transition to the proliferative phase has become a potential research direction in the treatment of diabetic chronic wounds. A novel snail glycosaminoglycan (AFG) extracted from the white jade snail (Achatina fulica var.) was found to significantly reduce early inflammatory responses and promote skin wound healing of diabetic rats in our preliminary study. To improve the convenience of drug delivery, a hydrogel named AFG/G30 hydrogel based on AFG and methacrylated gelatin (GelMA) was prepared by a photo-crosslinking method, with properties including easy of gelation, good tissue adhesion and biocompatibility. In this study, we evaluated the therapeutic effect of AFG/G30 hydrogel in a streptozotocin-induced diabetic wound rat model, and explored its mechanism of action in healing diabetic wounds. To assess the therapeutic efficacy of AFG/G30 hydrogel, we utilized saline, G30 and HA/G30 hydrogels as controls. The quantitative analysis of the healing progress on postoperative days 0, 3, 6, 10 and 14 showed that the group treated with AFG/G30 hydrogel outperformed the other groups. In addition, the results of H&E staining, Masson staining and α-SMA/CD31 immunofluorescence staining indicated that AFG/G30 hydrogel could effectively reduce inflammation and edema, and stimulate new blood vessels formation, epithelial regeneration and collagen deposition. Animal experiments showed that AFG/G30 hydrogel exerts significant anti-inflammatory effects and promotes diabetic wound healing. The mechanism of action of AFG/G30 hydrogel for healing diabetic wounds was explored initially through bioinformatics and molecular biology methods. Wound tissues on postoperative days 3, 10 were selected for transcriptome sequencing to identify differentially expressed genes and potential signaling pathways. A total of 1065 differentially expressed genes were screened, among which the number of differentially expressed genes in the AFG/G30 hydrogel group on day 3 was three times higher than the other two hydrogel groups, with 590 up-regulated genes and 593 down-regulated genes, and most of the down-regulated genes in G30 and HA/G30 hydrogel groups overlapped with those in the AFG/G30 group; GO/KEGG en
语种	中文
	2022-05
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75183
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	宋雪梅. 光交联的多糖AFG-GelMA水凝胶愈合糖尿病伤口的药效学研究[D]. 中国科学院大学,2022.

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