基于互穿网络凝胶构建小鼠胰岛仿生微环境的研究

Constructing biomimetic microenvironment for mouse islets based on interpenetrating network hydrogel

目的制备甲基丙烯酰化明胶/丝胶(GelMA/SS)互穿网络水凝胶支架,构建胰岛(Islets)仿生微环境。方法从6周龄美国癌症研究所(ICR)小鼠提取胰岛并经双硫腙染色鉴定。设置组织培养板(TCP)培养为对照组,单独GelMA以及不同质量比GelMA/SS(1∶1、1∶2、1∶4、1∶8)培养为实验组,分别记为G1S1、G1S2、G1S4和G1S8。扫描电镜下观察微观形态,流变学评价支架黏弹性,支架浸提液培养L929细胞评估生物相容性。钙黄绿素-乙酰甲氧基甲酯(AM)/碘化丙锭染色评价胰岛存活,活性氧(ROS)试剂盒评价拮抗ROS产生情况,酶联免疫吸附试验(ELISA)检测葡萄糖刺激胰岛素释放量。两组数据间比较采用t检验,多组数据组间比较采用方差分析。结果分离胰岛呈类圆形;水凝胶弹性随丝胶含量增加而下降,至GelMA/SS=1∶8时已不能成胶(G">G’);噻唑蓝(MTT)结果显示实验组均具有良好的生物相容性,且G1S4组显示出最高的细胞活力[(120.67±3.33)%]和显著拮抗ROS产生能力(5.18±0.48);活/死细胞染色显示实验组能够促进胰岛存活;葡萄糖刺激胰岛素释放(GSIS)结果显示在低糖刺激下G1S4具有最高的胰岛素分泌量[(7.73±0.52)μg/L],而高糖刺激下实验组与对照组差异无统计学意义(F=0.395,P>0.05)。结论GelMA/SS水凝胶具有良好的生物相容性和葡萄糖刺激响应能力,是构建仿生微环境,用于胰岛培养及移植的理想载体。

Objective Preparing the methacryloylated gelatin/sericin(GelMA/SS)interpenetrating network hydrogel scaffold for the construction of islets biomimetic microenvironment.Methods The islets were extracted from the pancreas of 6-week-old ICR mice and identified by dithizone staining.The islets were cultured on the tissue culture plate(TCP)as the control group,on GelMA and different mass ratio GelMA/SS(1∶1,1∶2,1∶4,1∶8)as the experimental groups,denoted as G1S1,G1S2,G1S4 and G1S8 groups respectively.Scanning electron microscopy was used to observe the microscopic morphology,rheology to evaluate the viscoelasticity of the scaffold,and the biocompatibility was evaluated by the scaffold extract to L929 cells.Calcein-AM/propidium iodide staining was done to evaluate islet survival,reactive oxygen species(ROS)kit was used to evaluate the antagonistic ROS production,and enzyme-linked immunosorbent assay(ELISA)kit was used to detect glucose-stimulated insulin release.T-test was used for comparison between two groups,and analysis of variance was used for comparison between multiple groups.Results The isolated islets were almost round.As the content of sericin increased,the elasticity of the hydrogel decreased,and gel could not formed when GelMA/SS=1∶8(G">G’).Methyl thiazolyl tetrazolium(MTT)results showed that all the experimental groups had good biocompatibility,and G1S4 group exhibited the highest cell viability[(120.67±3.33)%]and antagonistic ROS production(5.18±0.48).Live/dead cell staining showed that the experimental group could promote the survival of islets.The results of glucose-stimulated insulin release(GSIS)showed that G1S4 had the highest insulin secretion under low glucose stimulation[(7.73±0.52)μg/L],while under high glucose stimulation,no statistically significant difference existed between experimental group and control group(F=0.395,P>0.05).Conclusion GelMA/SS hydrogel exert good biocompatibility and glucose responsive ability,which would be an ideal carrier for constructing biomimetic microenvironment for islet culture and transplantation.