摘要
背景:构建仿生微环境促进胰岛素分泌细胞存活及功能发挥,是胰腺组织工程的热点与难点。目的:基于甲基丙烯酰化明胶/富血小板血浆水凝胶构建仿生微环境,促进小鼠胰岛瘤细胞MIN6存活和功能表达。方法:将体积分数10%,30%,50%的富血小板血浆溶液分别与50 g/L的甲基丙烯酰化明胶混合,经Ca^(2+)与凝血酶活化及紫外光照射固化成水凝胶,分别记为G+10P、G+30P、G+50P,同时制备单纯的甲基丙烯酰化明胶水凝胶(记为G),检测4组水凝胶的孔隙率、杨氏模量、溶胀性能与流变行为。将4组水凝胶分别与小鼠胰岛瘤细胞MIN6共培养,检测细胞形态与增殖活性,并进行qRT-PCR检测、胰岛素免疫荧光染色及胰岛素释放实验。结果与结论:①复合成分水凝胶的孔隙率小于单一成分水凝胶、杨氏模量高于单一成分水凝胶,并且随着富血小板血浆浓度的增加,复合成分水凝胶的孔隙率与杨氏模量降低;G+30P、G+50P组溶胀率低于G组、G+10P组(P<0.05);复合成分水凝胶的储能模量、耗能模量均大于单一成分水凝胶(P<0.05);②光镜下可见,单一成分水凝胶表面的细胞呈团块样且散在分布,复合成分水凝胶表面的细胞呈团状,但生长速度更快、细胞团之间连接紧密;活死染色显示,各组水凝胶可促进细胞存活,其中G+30P组、G+50P组死细胞数量明显少于G+10P组、G组;CCK-8检测显示,复合成分水凝胶促进细胞增殖效果强于单一成分水凝胶,并且随着富血小板血浆浓度的增加,促增殖效果更明显;③qRT-PCR检测显示,与单一成分水凝胶比较,复合成分水凝胶可明显上调胰岛十二指肠同源盒1、胰岛素、葡萄糖激酶的mRNA表达,其中以G+30P组最明显;免疫荧光与胰岛素释放实验显示,与单一成分水凝胶比较,复合成分水凝胶可促进胰岛素蛋白的表达及胰岛素释放;④结果表明,甲基丙烯酰化明胶/富血小板血浆复合水凝胶可用于模拟胰
BACKGROUND:In the field of pancreatic tissue engineering,constructing biomimetic microenvironment to promote the survival and functional exertion of insulin-secreting cells remains difficult point and hot problem.OBJECTIVE:To construct a biomimetic microenvironment based on gelatin methacrylamide/platelet-rich plasma hydrogel to promote the survival and cellular functional of MIN6.METHODS:10%,30%and 50%platelet-rich plasmas were mixed with 50 g/L final concentration of gelatin methacrylamide,gelation by the activation of Ca^(2+)/thrombin and ultraviolet irradiation(abbreviated as G+10P,G+30P,G+50P).Simultaneously,pure gelatin methacrylamide hydrogel was prepared(denoted as G).The porosity,Young’s modulus,swelling properties and rheological behavior of hydrogels of the four groups were tested.MIN6 cells were seeded on the gel surface,and cell morphology and proliferation were detected,followed by qRT-PCR,immunofluorescence and release assays of insulin.RESULTS AND CONCLUSION:(1)The porosity of the composite-component hydrogel was smaller than that of the single-component hydrogel,and the Young's modulus was higher than that of the single-component hydrogel.Furthermore,the porosity and Young's modulus of the composite hydrogel decreased with the increase of the platelet-rich plasma concentration.The swelling rate of the G+30P and G+50P groups was lower than that of the G and G+10P groups(P<0.05).The storage modulus and dissipation modulus of the composite hydrogels were higher than those of the single-component hydrogels(P<0.05).(2)Under the light microscope,the cells on the surface of the monocomponent hydrogel were clumpy and scattered;the cells on the surface of the compositecomponent hydrogel were clumps,but grew faster and the cell clusters were tightly connected.Live-dead staining showed that the hydrogels in each group could promote cell survival;the number of dead cells in G+30P and G+50P groups was significantly less than that in G+10P and G groups.The CCK-8 assay displayed that the composite-component h
作者
朱必文
王东芝
吴迪
龚天成
潘昊鹏
陆玉华
郭益冰
王志伟
黄䶮
Zhu Biwen;Wang Dongzhi;Wu Di;Gong Tiancheng;Pan Haopeng;Lu Yuhua;Guo Yibing;Wang Zhiwei;Huang Yan(First Department of Hepatic and Splenic Surgery,Affiliated Hospital of Nantong University,Nantong 226001,Jiangsu Province,China;Research Center of Clinical Medicine,Affiliated Hospital of Nantong University,Nantong 226001,Jiangsu Province,China;Medical School of Nantong University,Nantong 226001,Jiangsu Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2023年第12期1824-1831,共8页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(82001977),项目负责人:黄䶮
江苏省自然科学基金(BK20201445),项目负责人:黄䶮。
关键词
甲基丙烯酰化明胶
富血小板血浆
水凝胶
胰岛素
小鼠胰岛瘤细胞
仿生微环境
糖尿病
gelatin methacrylamide
platelet-rich plasma
hydrogel
insulin
mouse insulinoma cells
biomimetic microenvironment
diabetes mellitus
