中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (29): 4628-4633.doi: 10.12307/2024.583

• 水凝胶材料Hydrogel materials • 上一篇    下一篇

负载Scriptaid甲基丙烯酰化丝素蛋白水凝胶调控小胶质细胞的极化行为

郭青霞1，王  玥1，2，吴  桐1   

  1. 1青岛大学医学部，神经再生与康复研究院，山东省青岛市  266071；2青岛大学附属医院神经外科，山东省青岛市  266071
  • 收稿日期:2023-10-27 接受日期:2023-12-14 出版日期:2024-10-18 发布日期:2024-03-22
  • 通讯作者: 吴桐，博士，教授，硕士生导师，青岛大学医学部，神经再生与康复研究院，山东省青岛市 266071
  • 作者简介:郭青霞，女，1999年生，山东省威海市人，汉族，硕士，主要从事神经再生修复和组织工程方面的研究。
  • 基金资助:
    国家自然科学基金(32171322)，项目负责人：吴桐

Silk fibroin methacryloyl hydrogel loaded with Scriptaid regulates polarization of microglia cells

Guo Qingxia1, Wang Yue1, 2, Wu Tong1   

  1. 1Institute of Neuroregeneration and Neurorehabilitation, Medical College, Qingdao University, Qingdao 266071, Shandong Province, China; 2Department of Neurosurgery, Affiliated Hospital of Qingdao University, Qingdao 266071, Shandong Province, China
  • Received:2023-10-27 Accepted:2023-12-14 Online:2024-10-18 Published:2024-03-22
  • Contact: Wu Tong, PhD, Professor, Master’s supervisor, Institute of Neuroregeneration and Neurorehabilitation, Medical College, Qingdao University, Qingdao 266071, Shandong Province, China
  • About author:Guo Qingxia, Master, Institute of Neuroregeneration and Neurorehabilitation, Medical College, Qingdao University, Qingdao 266071, Shandong Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 32171322 (to WT)

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摘要：


文题释义：

Scriptaid：为一种组蛋白去乙酰化酶抑制剂，能够通过PTEN/PIP3/AKT通路调节小胶质细胞向M2型极化，具有抑制神经炎症和增强神经保护的作用。
小胶质细胞：是常驻于中枢神经系统的免疫效应细胞，可以极化为M1促炎表型和M2抗炎表型。M1型小胶质细胞分泌炎性因子和细胞毒性物质，有利于清除病原体，但是其过度激活会导致神经炎症，引起神经损伤；M2型小胶质细胞释放抗炎因子和神经营养因子，促进神经修复。


背景：小胶质细胞是维持中枢神经系统平衡的主要参与者，在神经系统发育和功能重建中起重要作用。Scriptaid作为一种组蛋白去乙酰化酶抑制剂具有抑制神经炎症和增强神经保护的作用。

目的：探索负载Scriptaid的甲基丙烯酰化丝素蛋白水凝胶对小胶质细胞行为的影响。
方法：采用含不同浓度(0，0.1，0.5，1，2，5，10 μmol/L)Scriptaid的培养液培养小胶质细胞(BV2细胞)，利用CCK-8法与活/死细胞染色筛选Scriptaid的适宜干预浓度。采用光固化制备负载与未负载Scriptaid(1 μmol/L)的甲基丙烯酰化丝素蛋白水凝胶，表征水凝胶的微观形貌、溶胀性能、力学性能、缓释性能及亲水性。将小胶质细胞(BV2细胞)接种于24孔Transwell小室下室，分5组培养：对照组将细胞培养液加入下室，脂多糖组、Scriptaid组、单纯水凝胶组、载药水凝胶组将含有脂多糖的细胞培养液加入下室，脂多糖干预24 h后，Scriptaid组、单纯水凝胶组、载药水凝胶组上室分别加入Scriptaid、甲基丙烯酰化丝素蛋白水凝胶、负载Scriptaid的甲基丙烯酰化丝素蛋白水凝胶，将培养液更换为普通培养液继续培养24 h，通过CCK-8法检测细胞活性，通过诱导型一氧化氮合酶和精氨酸酶1免疫荧光染色检测细胞表型。

结果与结论：①与未加入Scriptaid组相比，加入Scriptaid干预后BV2细胞的活性与数量均降低，当加入2 μmol/L 及以上Scriptaid时细胞活性低于标准化细胞活性(70%)，并且细胞数量显著减少，所以选择1 μmol/L Scriptaid负载于水凝胶中；②表征实验显示，Scriptaid的加入未影响甲基丙烯酰化丝素蛋白水凝胶的微观形貌、吸水溶胀率、压缩模量与亲水性，甲基丙烯酰化丝素蛋白水凝胶具有缓释性能；③CCK-8法检测显示，与对照组相比，甲基丙烯酰化丝素蛋白水凝胶显著提高了BV2细胞活性(P < 0.001)；④免疫荧光染色显示，与对照组相比，脂多糖组细胞诱导型一氧化氮合酶表达量增加(P < 0.01)，载药水凝胶组细胞诱导型一氧化氮合酶表达量减少(P < 0.01)、精氨酸酶1表达量增加(P < 0.001)，Scriptaid组细胞精氨酸酶1表达量增加(P < 0.01)；⑤结果表明，负载Scriptaid的甲基丙烯酰化丝素蛋白水凝胶可促进脂多糖诱导后的小胶质细胞向M2型极化。

https://orcid.org/0009-0000-0180-3970(郭青霞)

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: Scriptaid, 甲基丙烯酰化丝素蛋白, 水凝胶, 小胶质细胞, 细胞极化

Abstract: BACKGROUND: Microglia cells play a major role in maintaining the balance as well as the development and function reconstruction of the central nervous system. As a histone deacetylase inhibitor, Scriptaid can inhibit neuroinflammation and enhance neuroprotection.
OBJECTIVE: To investigate the effect of silk fibroin methacryloyl hydrogel loaded with Scriptaid on the behaviors of microglia cells.
METHODS: Microglia (BV2 cells) were cultured in medium containing different concentrations of Scriptaid (0, 0.1, 0.5, 1, 2, 5, 10 μmol/L). The optimal concentration of Scriptaid was screened by the CCK-8 assay and live/dead cell staining. Silk fibroin methacryloyl hydrogels loaded with or without Scriptaid were prepared using photocuring. The micromorphology, swelling properties, mechanical properties, slow release properties, and hydrophilicity of the hydrogels were characterized. Microglia (BV2 cells) were inoculated in the subventricular region of 24-well Transwell and cultured in five groups. In the control group, the cell culture medium was added to the lower chamber. In the lipopolysaccharide group, Scriptaid group, hydrogel group, and drug-loaded hydrogel group, cell culture media containing lipopolysaccharide were added into the lower chamber. After 24 hours of lipopolysaccharide intervention, in the Scriptaid group, hydrogel group and drug-loaded hydrogel group, Scriptaid, silk fibroin methacryloyl hydrogel, and silk fibroin methacryloyl hydrogel loaded with Scriptaid were added to the upper chamber, respectively. The culture medium was replaced with ordinary culture medium and continued to culture for 24 hours. The cell viability was detected by CCK-8 assay, and the cell phenotype was detected by immunofluorescence staining of induced nitric oxide synthase and arginase 1.
RESULTS AND CONCLUSION: (1) Compared with the group without Scriptaid, the viability and number of BV2 cells were decreased after Scriptaid added. When Scriptaid 2 μmol/L or above was added, the cell viability was lower than the standardized cell viability (70%), and the number of BV2 cells was significantly reduced. Therefore, 1 μmol/L Scriptaid was selected to be loaded into the hydrogel. (2) Characterization experiments showed that the addition of Scriptaid did not affect the microscopic morphology, swelling rate of water absorption, compression modulus and hydrophilicity of silk fibroin methacryloyl hydrogel, and silk fibroin methacryloyl hydrogel had slow release performance. (3) The result of CCK-8 assay showed that compared with the control group, silk fibroin methacryloyl hydrogel significantly increased the viability of BV2 cells (P < 0.001). (4) Immunofluorescence staining showed that compared with the control group, the expression of inducible nitric oxide synthase was increased in the lipopolysaccharide group (P < 0.01); the expression of inducible nitric oxide synthase was decreased (P < 0.01) and the expression of arginase 1 was increased (P < 0.001) in the drug-loaded hydrogel group; the expression of arginase 1 was increased in the Scriptaid group (P < 0.01). (5) The results indicate that Scriptaid-loaded silk fibroin methacryloyl hydrogel is able to promote polarization of microglia to the M2 type after lipopolysaccharide induction. 

Key words: Scriptaid, silk fibroin methacryloyl, hydrogel, microglia, cell polarization

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