壳寡糖构建的胰岛仿生微环境通过降低细胞内活性氧保护胰岛免受低氧诱导损伤

摘要：目的 制备甲基丙烯酰化明胶（gelatin methacryloyl,GelMA）/甲基丙烯酰化透明质酸（hyaluronic acid methacryloyl,HAMA）/壳寡糖（chitosan oligosaccharide,COS）水凝胶，用于构建胰岛仿生微环境，并探讨GelMA/HAMA/COS水凝胶对低氧下胰岛活性和功能的改善作用。

方法：以组织培养板培养为对照组，COS浓度分别为0、1、5、10、20 mg/mL的GelMA/HAMA/COS水凝胶为实验组，扫描电镜观察微观形态，流变仪评价成胶性能，接触角检测亲水性，水凝胶浸提液培养L929细胞评估生物相容性[采用细胞计数试剂盒8（cell counting kit8,CCK-8）法]。从8周龄SD大鼠胰腺中提取胰岛，双硫腙染色和葡萄糖刺激胰岛素释放（glucose-stimulated insulin secretion,GSIS）实验分别鉴定胰岛纯度和功能。

胰岛在低氧（1%O2）下分别培养24、48、72 h，使用钙黄绿素-乙酰甲氧基甲酯/碘化丙啶（Calcein-acetyl methyl/propidium iodide,Calcein-AM/PI）染色评价低氧对胰岛活性的影响。将胰岛在不同COS浓度的GelMA/HAMA/COS水凝胶中培养48 h，活性氧试剂盒评价COS在常氧（20%O2）和低氧（1%O2）条件下拮抗胰岛活性氧产生情况，Calcein-AM/PI染色评价COS在低氧（1%O2）条件下对胰岛活性的影响。将胰岛分别在组织培养板（A组）、GelMA/HAMA水凝胶（B组）和GelMA/HAMA/COS水凝胶（C组）中培养48 h，免疫荧光和GSIS实验分别评价COS在低氧（1%O2）条件下对胰岛活性的影响。结果 GelMA/HAMA/COS水凝胶呈多孔结构，流变仪检测提示其具有较好的成胶性能，接触角表现出良好亲水性，CCK-8法检测示各组水凝胶均具有良好的生物相容性。分离的大鼠胰岛呈类圆形，具有较高的胰岛纯度和胰岛素分泌能力。

胰岛在低氧条件下处理24、48、72 h,Calcein-AM/PI染色示死细胞比例随时间延长逐渐增多，均显著高于未低氧处理组（P<0.001）。活性氧染色示不同COS浓度的GelMA/HAMA/COS水凝胶在常氧和低氧条件下均能拮抗活性氧的产生，且该能力与COS浓度成正相关。Calcein-AM/PI染色示，不同COS浓度的GelMA/HAMA/COS水凝胶在低氧条件下均能改善胰岛活性，细胞活性与COS浓度成正相关。免疫荧光染色示GelMA/HAMA/COS水凝胶在低氧条件下能促进胰岛功能相关基因的表达；GSIS实验结果显示C组胰岛在低氧条件胰岛素分泌量显著高于A、B组（P<0.05）。

结论： GelMA/HAMA/COS水凝胶具有良好的生物相容性，通过抑制活性氧提高胰岛的存活和功能，是构建胰岛仿生微环境用于胰岛培养及移植的理想载体。

• [1]Protective Effect of Chitosan Oligosaccharide against Hydrogen Peroxide-Mediated Oxidative Damage and Cell Apoptosis via Activating Nrf2/ARE Signaling Pathway.[J] . Zhang Xiaoxia,Liang Shuang,Gao Xiaohan,Huang Hanchang,Lao Fengxue,Dai Xueling.  Neurotoxicity research . 2021

• [2]Engineering a Macroporous Oxygen-generating Scaffold for Enhancing Islet Cell Transplantation within an Extrahepatic Site.[J] . Liang JiaPu,Accolla Robert P,Soundirarajan Madhuvanthi,Emerson Amy,Coronel Maria M,Stabler Cherie.  Acta biomaterialia . 2021

• [3]Preservation of pancreas in the University of Wisconsin solution supplemented with AP39 reduces reactive oxygen species production and improves islet graft function.[J] . Nishime Kai,MiyagiShiohira Chika,Kuwae Kazuho,Tamaki Yoshihito,Yonaha Tasuku,SakaiYonaha Mayuko,Saitoh Issei,Watanabe Masami,Noguchi Hirofumi.  American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons . 2020 (8)

• [4]Hybrid Printing Using Cellulose Nanocrystals Reinforced GelMA/HAMA Hydrogels for Improved Structural Integration.[J] . Fan Yuchao,Yue Zhilian,Lucarelli Enrico,Wallace Gordon G.  Advanced healthcare materials . 2020

• [5]Reactive oxygen species-responsive silk sericin microcapsules used for antioxidative stress damage.[J] . Wang Guangshu,Wang Zhenyu,Sun Ning,Cai Yurong,Yang Xiaogang.  Microscopy research and technique . 2020 (4)

• [6]Immune-evasive human islet-like organoids ameliorate diabetes.[J] . Yoshihara Eiji,O’Connor Carolyn,Gasser Emanuel,Wei Zong,Oh Tae Gyu,Tseng Tiffany W,Wang Dan,Cayabyab Fritz,Dai Yang,Yu Ruth T,Liddle Christopher,Atkins Annette R,Downes Michael,Evans Ronald M.  Nature . 2020

• [7]Significant reduction of apoptosis induced via hypoxia and oxidative stress in isolated human islet by resveratrol[J] . Somayeh Keshtkar,Maryam Kaviani,Zahra Jabbarpour,Ismail H. Al- Abdullah,Mahdokht H. Aghdaei,Saman Nikeghbalian,Alireza Shamsaeefar,Bita Geramizadeh,Negar Azarpira,Mohammad H. Ghahremani.  Nutrition, Metabolism and Cardiovascular Diseases . 2020 (7)

• [8]A Collagen Based Cryogel Bioscaffold that Generates Oxygen for Islet Transplantation[J] . Mehdi Razavi,Rosita Primavera,Bhavesh D. Kevadiya,Jing Wang,Peter Buchwald,Avnesh S. Thakor.  Advanced Functional Materials . 2020 (15)

• [9]Bio-ink development for three-dimensional bioprinting of hetero-cellular cartilage constructs.[J] . Mouser Vivian H M,Levato Riccardo,Mensinga Anneloes,Dhert Wouter J A,Gawlitta Debby,Malda Jos.  Connective tissue research . 2020 (2)

• [10]Human mesenchymal stem cells-derived conditioned medium inhibits hypoxia-induced death of neonatal porcine islets by inducing autophagy.[J] . Xu Yuzhi,Tan Mengqun,Ma Xiaoqian,Li Hongde,He Xuesong,Chen Zeyi,Tan Yixiong,Nie Wei,Rong Pengfei,Wang Wei.  Xenotransplantation . 2020 (1)