【作者】 周亮；

【导师】 张艳；

【作者基本信息】 南京大学 ， 化学生物学， 2021， 硕士

【摘要】 光照作为一种具有高时空分辨率的外界刺激方式,具有优良的生物相容性,因此能够以无损的方式对生物体系进行时空分辨的动态调控。近年来,光驱动的生物正交反应逐步被开发,例如紫外光诱导的四氮唑与缺电子烯烃(TEPC)间的点击反应以及可见光诱导的菲醌与烯醚(DVPC)间的点击反应。使得基于光驱动的成键反应对生物体系的修饰成为可能。本论文基于TEPC和DVPC两种生物正交反应在短肽自组装体系修饰以及蛋白结构的修饰方面开展了研究。论文第一部分基于四氮唑(TPI)修饰的短肽TPI-GGF及菲醌(PQ)修饰的短肽PQ-GFF构建的双组分超分子水凝胶开展研究,用光谱对水凝胶基质中的纳米纤维进行表征。进而基于TPI与富马酸单甲酯(MF)的TEPC光点击反应及PQ与烯醚(VE)间的DVPC光点击反应,探索了在双组分水凝胶中通过紫外及可见两种不同波长的光调控在自组装纳米纤维上共价粘附不同分子的可能性,为基于光驱动生物正交反应对水凝胶基质进行动态修饰,以构建能受光照动态调控的三维细胞微环境奠定了基础。论文第二部分研究了三维培养在TPI-GGF与PQ-GFF双组分水凝胶中的干细胞随微环境变化而产生的行为变化。借助TPI与MF间的TEPC反应及PQ与VE间的DVPC反应,将混合在水凝胶基质中的MF-GGGHAV促软骨分化功能短肽及促细胞粘附的VE-c(RGDf K)短肽可控粘附到凝胶基质中不同的纳米纤维上。论文第三部分对DVPC反应产物PDO在力诱导作用下是否可以断键进行了探索。利用SNAP-tag技术我们将BG-PDO-BG(BPB)与(GB1)4-SNAP蛋白相结合构建了SNAP-PDO-SNAP(SPS)蛋白聚合物,超声初步结果表明蛋白聚合物中BPB位置发生断键,但是否按照设想断裂成原来菲醌和烯醚的结构还在进一步验证当中。综上所述,论文主要围绕着可见光诱导的菲醌与烯醚间的生物正交反应(DVPC)的生物学应用探索展开,TPI-GGF与PQ-GFF双组分水凝胶的成功构建用于研究干细胞对微环境生物要素变化的行为响应机制研究提供了新方向。DVPC光点击反应产物PDO的超声断键研究仍在进行中。更多还原

【Abstract】 As an external stimulus with high spatial and temporal resolution,illumination has excellent biocompatibility,so it can dynamically regulate the spatial and temporal resolution of biological systems in a nondestructive way.In recent years,light-driven bioorthogonal reactions have been gradually developed,such as the UV-induced click reaction between tetrazole and electron-deficient olefin(TEPC)and visible lightinduced click reaction between phenanthraquinone and vinyl ether(DVPC).This makes it possible to modify biological systems based on light-triggered biorthogonal ligation reactions.In this thesis,we studied the modification of short peptide self-assembly system and protein structure based on TEPC and DVPC.In the first part of this thesis,a two-component supramolecular hydrogel was constructed based on tetrazole(TPI)modified short peptide TPI-GGF and phenanthraquinone(PQ)modified short peptide PQ-GFF,and the nanofibers in the hydrogel matrix were characterized by spectrum.Then,based on the TEPC photo-click reaction between TPI and monomethyl fumarate(MF)and the DVPC photo-click reaction between PQ and vinyl ether(VE),the possibility of covalently adhering to different molecules on self-assembled nanofibers was explored in the two-component hydrogel by ultraviolet and visible light regulation of different wavelengths.This study lays a foundation for the dynamic modification of hydrogel matrix based on the lightdriven bioorthogonal reaction to construct a three-dimensional cell microenvironment which can be dynamically regulated by light.In the second part,we studied the behavior of stem cells cultured in TPI-GGF and PQ-GFF two-component hydrogel with the change of microenvironment.By means of TEPC reaction between TPI and MF and DVPC reaction between PQ and VE,MFGGGHAV short peptide and VE-c(RGDf K)short peptide mixed in the hydrogel matrix were controlled to adhere to different nanofibers in the gel matrix.In the third part of the thesis,whether PDO,the reaction product of DVPC,can break the bond under the force is explored.Using snap-tag technology,we combined BG-PDO-BG(BPB)with(GB1)4-SNAP protein to construct SNAP-PDO-SNAP(SPS)protein polymer.Preliminary ultrasonic results showed that the BPB position in the protein polymer was broken.However,whether to fracture into the original phenanthraquinone and vinyl ether is still in process.In conclusion,this thesis mainly focuses on the biological application exploration of visible light-induced bioorthogonal reaction between phenanthraquinone and vinyl ether(DVPC).The successful construction of TPI-GGF and PQ-GFF two-component hydrogel provides a new direction for the study of the behavioral response mechanism of stem cells to the changes of biological elements in the microenvironment.The study of ultrasonic bond breaking of PDO,the product of DVPC photoclick reaction,is still in progress.更多还原