具膜锚定性能的双功能生长因子模拟物构建及其在类器官培养中的应用

Construction of a membrane-anchored bifunctional growth factor mimetic and its application in organoid culture

摘要:

类器官模型作为一种新型的活体组织离体培养模型(ex vivo model)，助力了生物医学研究与生物医药开发，然而类器官培养中所需重组生长因子成本高昂、批次均一性低、稳定性差等问题限制了类器官模型的标准化与规模化应用。本研究旨在构建一种基于功能核酸的膜锚定双功能生长因子模拟物，以替代重组生长因子用于类器官培养。采用核酸适体作为生长因子功能的模拟单元，并以胆固醇(cholesterol, Chol)作为膜锚定模块，利用碱基互补配对原则构建了2种双功能生长因子模拟物：其一为靶向结合诱导表皮生长因子受体(epidermal growth factor receptor, EGFR)与成纤维细胞生长因子受体1 (fibroblast growth factor receptor 1, FGFR1)二聚激活的双特异性核酸适体(ApE+F-Chol)，旨在模拟表皮生长因子(epidermal growth factor, EGF)与碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)的协同功能；其二为设计可靶向并诱导肝细胞生长因子受体(mesenchymal-epithelial transition factor, Met)与FGFR1二聚激活的双特异性核酸适体(ApF+M-Chol)，以模拟肝细胞生长因子(hepatocyte growth factor, HGF)与bFGF的功能。结果显示，互补序列为22个碱基的膜锚定双功能生长因子——ApE+F-22-Chol能够成功锚定于细胞膜，具有最佳类EGF与bFGF性能，可同时高效激活EGFR与FGFR1通路及下游信号通路，在功能上可协同促进细胞的增殖与迁移；在患者来源前列腺癌类器官培养体系中，ApE+F-22-Chol可完全替代培养液中的EGF和bFGF重组因子，有效促进类器官的形成与生长。此外，基于相同策略构建的ApF+M-Chol可以同时模拟HGF与bFGF功能，同样在类器官培养中展现出显著活性，验证了该膜锚定双功能生长因子模拟物构建策略具有良好的通用性。本研究为类器官培养体系提供了成分明确、稳定且高效的新型工具，在疾病建模、药物筛选及再生医学等领域具有广阔的应用前景。

Abstract:

Currently, organoids emerges as novel ex vivo models for biomedical research and biopharmaceutical development. However, the recombinant growth factors used for organoid culture faces challenges such as high costs, low batch consistency, and poor stability, which limit the standardization and scaling of organoid applications. This study aims to develop a membrane-anchored bifunctional growth factor mimetic based on functional nucleic acids to replace recombinant growth factors in organoid culture. Using nucleic acid aptamers as functional mimic units and cholesterol (Chol) as the membrane anchoring module, we constructed two bifunctional growth factor mimetics through base-complement pairing principles. The first was a dual-specificity aptamer (ApE+F-22-Chol) targeting the dimerization activation of both epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1), simulating the synergistic function of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). The second was a dual-specificity aptamer (ApF+M-Chol) designed to target and induce the dimerization activation of mesenchymal-epithelial transition factor (Met) and FGFR1, mimicking the function of hepatocyte growth factor (HGF) and bFGF. The results demonstrated that ApE+F-22-Chol successfully anchored to cell membranes while efficiently activating both EGFR and FGFR1 signaling pathways, synergistically promoting cell proliferation and migration. In a patient-derived prostate cancer organoid culture system, ApE+F-22-Chol can fully replace EGF and bFGF in the culture medium, effectively supporting organoid formation and growth. Further experiments confirmed that the ApF+M-Chol constructed with the same strategy also exhibited significant growth-promoting activity in organoid culture, validating the broad applicability of this membrane-anchored bifunctional growth factor mimetic strategy. This study provides a novel tool with well-defined components, high stability, and high efficiency for organoid culture systems, showing promising applications in disease modeling, drug screening, and regenerative medicine.