归芪多糖基水凝胶的制备及其与3D喷墨打印的血管内皮细胞互作机理研究

3D cell printing is expected to solve the problem of vascularization of thick three-dimensional engineered tissue constructs. While cells trauma in printing suppress rapid vascularization, as can be improved by wellbehaved bioink. But currently available bioink materials isn't able to satisfy the requirement. Cytokines added in the hydrogel degrade too rapidly to meet the demand. So new types of functional materials which can protect vascular endothelial cells，promote vascularization and possess low degradation rate are needed. It is reported and proved by our early study, that guiqi polysaccharides, polysaccharides extracted from complex prescription of genunie medicinal material of Gansu province, angelica sinensis and radix astragali, have cytoprotection and vascularization promoting function. While they have not been used as bioink material, and the mechanism they protect cells and promote vascularization kept unknown. So in this project, we are going to prepare hydrogel based on guiqi polysaccharides, make blood vessels with 3D inkjet printer, observe cellular morphology of vascular endothelial cells in the bioink and degradation of the hydrogel, find influence of guiqi polysaccharides on vascular endothelial cells’ protein expression profile, then analyze the critical signal transduction pathway by western blot, probe into interaction between hydrogel based on guiqi polysaccharides and 3D inkjet printed vascular endothelial cells in molecular level. It will lay the foundation for development and screening of a new types of bioink materials with durative cytoprotection and vascularization promoting function, and gives us a new way to solve the problem of western medicine with chinese medicine at the same time.

三维细胞打印有望解决组织工程器官内血管网络形成的难题，但打印过程对血管内皮细胞造成损伤，这就要求“生物墨汁”提供的微环境具有持久的保护细胞和促血管化作用，然而目前的“生物墨汁”在活性、安全性、作用时间等方面不能完全满足要求。已有的报道和我们的研究结果表明，中药成分归芪多糖有较好的保护细胞和促进血管化功能，且作用时间长，但尚未见用于细胞打印的报道，其作用机理也不清楚，因此本项目拟用归芪多糖制备水凝胶，与细胞混合后通过喷墨打印制备人工血管模型，体外培养和体内移植观察血管内皮细胞的形态变化，分析细胞对水凝胶的降解规律，用荧光差异双向凝胶电泳研究归芪多糖对血管内皮细胞蛋白表达的影响，用western杂交、定量PCR等技术分析关键蛋白因子的信号通路，从细胞和分子水平探究归芪多糖的作用机理，为开发和筛选具有持久保护血管内皮细胞和促血管化作用的“生物墨汁”材料奠定基础,也为中医药解决西医难题提供新思。

载细胞3D打印有望解决组织工程器官内血管网络形成的难题，但打印过程对血管内皮细胞造成损伤，中药成分归芪多糖有较好的保护细胞和促进血管化功能，且作用时间长，但尚未见用于细胞打印的报道，其作用机理也不清楚，因此本项目对归芪多糖活性组分分离、筛选和评价，以其制备水凝胶，用喷墨打印制备人工血管，对其体内外细胞保护及促血管化活性进行了分析，并用荧光差异双向凝胶电泳初步研究了其作用的分子机理。. 多糖组分分离及活性研究显示，粗多糖细胞保护及促血管化效果优于单独组分，可能由于各多糖组分间存在协同作用所致，故后续试验采用粗多糖。复合多糖可以通过与壳聚糖交联制备水凝胶，可通过3D打印形成并保持血管形状，载血管内皮细胞的支架显示出细胞保护和促内皮细胞增殖活性。荧光差异双向凝胶电泳结果显示，归芪多糖凝胶组作用于血管内皮细胞后，表达量上调1.2倍以上的蛋白点有46个，表达量下调1.2倍以上有29个。通过软件分析筛选其中27个蛋白点作MALDI-TOF-MS。通过western杂交验证归芪多糖水凝胶体外对血管内皮细胞的影响，显示其可能通过VEGF-MAPK途径促进细胞增殖，通过E钙黏素（E-cadherin）促进细胞之间的黏着，通过钙/钙调素依赖的丝氨酸激酶(CASK)促进细胞的形变和迁移；从而促进血管生成。小鼠体内实验进一步证实，归芪多糖水凝胶在体内移植后通过VEGF-MAPK信号途径和E-cadherin-CASK信号途径促进内皮细胞血管化，通过Id1-P16信号途径保护内皮细胞在热激诱导下不受损伤。. 本项目证实复方多糖可制备成可用于3D打印的水凝胶，并能保持其生物学活性，制备载细胞血管支架，发挥其细胞保护及促血管化作用。这些结果对于中医药的现代化应用具有重要意义。