中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (12): 1824-1831.doi: 10.12307/2023.082

• 药物控释材料 drug delivery materials • 上一篇    下一篇

甲基丙烯酰化明胶/富血小板血浆复合水凝胶构建仿生微环境促进小鼠胰岛瘤细胞MIN6功能

朱必文1，2，3，王东芝1，2，3，吴  迪1，2，3，龚天成2，3，潘昊鹏2，陆玉华1，2，3，郭益冰2，3，王志伟1，2，3，黄  䶮1，2，3   

  1. 南通大学附属医院，1肝胆胰脾外科一，2临床医学研究中心，江苏省南通市  226001；3南通大学医学院，江苏省南通市  226001
  • 收稿日期:2021-12-24 接受日期:2022-02-18 出版日期:2023-04-28 发布日期:2022-07-30
  • 通讯作者: 黄䶮，博士，主治医师，南通大学附属医院，肝胆胰脾外科一，临床医学研究中心，江苏省南通市 226001；南通大学医学院，江苏省南通市 226001 王志伟，教授，主任医师，博士生导师，南通大学附属医院，肝胆胰脾外科一，临床医学研究中心，江苏省南通市 226001；南通大学医学院，江苏省南通市 226001
  • 作者简介:朱必文，男，1996年生，江苏省盐城市人，汉族，南通大学在读硕士，主要从事生物材料在类胰岛构建中的应用研究。
  • 基金资助:
    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

Biomimetic microenvironment constructed from gelatin methacrylamide/platelet-rich plasma hydrogel promotes the function of insulinoma cell line MIN6 in mice

Zhu Biwen1, 2, 3, Wang Dongzhi1, 2, 3, Wu Di1, 2, 3, Gong Tiancheng2, 3, Pan Haopeng2, Lu Yuhua1, 2, 3, Guo Yibing2, 3, Wang Zhiwei1, 2, 3, Huang Yan1, 2, 3   

  1. 1First Department of Hepatic and Splenic Surgery, 2Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China; 3Medical School of Nantong University, Nantong 226001, Jiangsu Province, China
  • Received:2021-12-24 Accepted:2022-02-18 Online:2023-04-28 Published:2022-07-30
  • Contact: Huang Yan, MD, Attending physician, First Department of Hepatic and Splenic Surgery, and 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 Wang Zhiwei, Professor, Chief physician, Doctoral supervisor, First Department of Hepatic and Splenic Surgery, and 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
  • About author:Zhu Biwen, Master candidate, First Department of Hepatic and Splenic Surgery, and 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
  • Supported by:
    the National Natural Science Foundation of China, No. 82001977 (to HY); the Natural Science Foundation of Jiangsu Province, No. BK20201445 (to HY)

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摘要：

文题释义：
甲基丙烯酰化明胶：由明胶经甲基丙烯酰化制备而成，具有良好的理化性能和生物相容性，在细胞培养、组织工程等领域具有广泛应用，可通过模拟细胞外基质构建细胞仿生微环境，促进细胞的黏附、存活和功能发挥。
富血小板血浆：经梯度离心制备，血小板含量较全血高2-8倍，由Ca2+/凝血酶激活后释放大量具有独特促细胞增殖和分化功能的生长因子及细胞因子，主要包括血小板源性生长因子、转化生长因子β、胰岛素样生长因子、血管内皮生长因子等，目前主要应用于肌肉再生、创面修复等方面，效果显著。

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Ca2+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岛素分泌细胞微环境，能够显著提高其存活和功能发挥。

https://orcid.org/0000-0003-2725-0360 (朱必文)

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料；口腔生物材料；纳米材料；缓释材料；材料相容性；组织工程

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

Abstract: 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 Ca2+/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 composite-component 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 hydrogel had a stronger promoting effect on cell proliferation than the monocomponent hydrogel. Moreover, with the increase of platelet-rich plasma concentration, the promoting effect on proliferation was more obvious. (3) qRT-PCR demonstrated that compared with the monocomponent hydrogel, the composite-component hydrogel could significantly up-regulate the mRNA expression levels of islet-duodenum homeobox 1, insulin, and glucokinase; among them, the G+30P group was the most obvious. Immunofluorescence and insulin release assays exhibited that compared with monocomponent hydrogels, composite-component hydrogels could promote insulin protein expression and insulin release. (4) The results confirm that the gelatin methacrylamide/platelet-rich plasma hydrogel can be used to simulate the microenvironment of insulin-secreting cells and can significantly improve their survivals and functions.  

Key words: gelatin methacrylamide, platelet-rich plasma, hydrogel, insulin, mouse insulinoma cells, biomimetic microenvironment, diabetes mellitus

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