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  

Islet biomimetic microenvironment constructed by chitosan oligosaccharide protects islets from hypoxia-induced damage by reducing intracellular reactive oxygen species

作　　者：王东芝[1,2] 郭益冰[2] 黄龚 朱必文 潘浩鹏 王志伟[1] WANG Dongzhi;GUO Yibing;HUANG Yan;ZHU Biwen;PAN Haopeng;WANG Zhiwei(Department of General Surgery,Affiliated Hospital of Nantong University,Nantong Jiangsu,226001,P.R.China;Research Center of Clinical Medicine,Affiliated Hospital of Nantong University,Nantong Jiangsu,226001,P.R.China;Key Laboratory of Neuroregeneration,Nantong University,Nantong Jiangsu,226001,P.R.China)

机构地区：[1]南通大学附属医院普外科,江苏南通226001 [2]南通大学附属医院临床医学研究中心,江苏南通226001 [3]南通大学神经再生重点实验室,江苏南通226001

出　　处：《中国修复重建外科杂志》2022年第5期633-642,共10页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学青年科学基金(82001977);江苏省研究生科研与实践创新计划项目(KYCX19_2073)。

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%O_(2))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%O_(2))和低氧(1%O_(2))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%O_(2))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%O_(2))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Objective Gelatin methacryloyl(GelMA)/hyaluronic acid methacryloyl(HAMA)/chitosan oligosaccharide(COS)hydrogel was used to construct islet biomimetic microenvironment,and to explore the improvement effect of GelMA/HAMA/COS on islet activity and function under hypoxia.Methods Islets cultured on the tissue culture plate was set as the control group,on the GelMA/HAMA/COS hydrogel with COS concentrations of 0,1,5,10,and 20 mg/mL respectively as the experimental groups.Scanning electron microscopy was used to observe the microscopic morphology,rheometer test to evaluate the gel-forming properties,contact angle to detect the hydrophilicity,and the biocompatibility was evaluated by the scaffold extract to L929 cells[using cell counting kit 8(CCK-8)assay].The islets were extracted from the pancreas of 8-week-old Sprague Dawley rats and the islet purity and function were identified by dithizone staining and glucose-stimulated insulin secretion(GSIS)assays,respectively.Islets were cultured under hypoxia(1%O_(2))for 24,48,and 72 hours,respectively.Calcein-acetyl methyl/propidium iodide(Calcein-AM/PI)staining was used to evaluate the effect of hypoxia on islet viability.Islets were cultured in GelMA/HAMA/COS hydrogels with different COS concentrations for 48 hours,and the reactive oxygen species kits were used to evaluate the antagonism of COS against islet reactive oxygen species production under normoxia(20%O_(2))and hypoxia(1%O_(2))conditions.CalceinAM/PI staining was used to evaluate the effect of COS on islet activity under hypoxia(1%O_(2))conditions.Islets were cultured in tissue culture plates(group A),GelMA/HAMA hydrogels(group B),and GelMA/HAMA/COS hydrogels(group C)for48 hours,respectively.Immunofluorescence and GSIS assays were used to evaluate the effect of COS on islet activity under hypoxia(1%O_(2))conditions,respectively.Results GelMA/HAMA/COS hydrogel had a porous structure,the rheometer test showed that it had good gel-forming properties,and the contact angle test showed good hydrophilicity.CCK-8 assay showe

关 键 词：胰岛 壳寡糖 活性氧 仿生微环境 

分 类 号：R657.5[医药卫生—外科学]