| 赵倩,袁国庆,赵裕擎,等.基于岩藻糖基转移酶的HAase-NK92-MI细胞偶联物的构建及增强实体瘤渗透的研究[D][J].中国海洋药物,2026,(4):-. |
| 基于岩藻糖基转移酶的HAase-NK92-MI细胞偶联物的构建及增强实体瘤渗透的研究[D] |
| Construction of? hyaluronidase-NK92-MI cell conjugate by fucosyltransferase and its enhancement of solid tumor penetration |
| 投稿时间：2024-11-18 修订日期：2025-04-25 |
| DOI： |
| 中文关键词: HAase 岩藻糖基化 NK-92MI细胞 点击化学反应 |
| English Keywords:hyaluronidase fucosylation NK92-MI cells click chemistry |
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| 中文摘要: |
| 目的 利用α-1,3岩藻糖基转移酶，将海洋细菌来源的透明质酸酶（HAase）偶联到NK-92MI细胞上，构建HAase-NK-92MI细胞偶联物，使偶联细胞获得依赖HAase的肿瘤细胞外基质降解能力。方法 首先通过大肠杆菌分选酶Sortase催化的转肽反应，将携带有六肽标签的HAase与带有末端甘氨酸和click反应基团的小分子linker相连，随后通过点击化学与GDP-岩藻糖反应制备GDP-fucose-HAase，接下来通过α-1,3岩藻糖基转移酶催化的转糖基反应将其转移到NK-92MI细胞表面糖链上。利用流式细胞术及共聚焦成像技术，评价HAase-NK-92MI细胞偶联物是否成功构建；通过细胞增殖检测和Transwell侵袭实验评价偶联细胞的活性和细胞外基质降解能力；最后在肿瘤异种移植小鼠中评价偶联细胞的肿瘤浸润能力。结果 成功在岩藻糖基转移酶的作用下将HAase转移到NK-92MI细胞表面糖链上，构建了HAase-NK-92MI细胞偶联物。并且在体外和体内实验中，验证了该细胞偶联物具有良好的降解细胞外基质的能力和对肿瘤组织的渗透性，改善了实体瘤的免疫治疗效果。结论 通过基于岩藻糖基转移酶的偶联方案，成功在NK-92MI细胞表面引入HAase，提高了免疫细胞对肿瘤部位的浸润，从而增强了对实体瘤的渗透性，为探索新一代NK细胞的肿瘤免疫疗法提供了新的方案。 |
| English Summary: |
| Objective To construct a HAase-NK-92MI cell conjugate by coupling the hyaluronidase (HAase) of marine bacterium origin to NK-92MI cells using α-1,3 fucosyltransferase, which enabled the conjugate to acquire tumor extracellular matrix degradation ability. Methods Firstly, HAase carrying a hexapeptide tag was linked to a small molecule linker with terminal glycine and click-reactive motifs by a transpeptidation reaction catalyzed by the Escherichia coli sortase, followed by the preparation of GDP-fucose-HAase by the reaction of GDP-fucose via click chemistry, and next it was transferred to NK-92MI cells by the transglycosylation reaction catalyzed by α-1,3-fucosyltransferase to construct HAase-NK-92MI cell conjugate, which was verified by fluorescence imaging and flow cytometry, and the cell viability and extracellular matrix degradation of the conjugate were evaluated by CCK8 assay and transwell invasion assay; finally, the tumor infiltration capacity of HAase-NK-92MI cell conjugate were evaluated in tumor xenograft mice. Results The HAase-NK-92MI cell conjugates was constructed successfully by transferring hyaluronidase to NK-92MI cell surface via fucosyltransferase, and it was verified in vitro and in vivo experiments that the conjugates had a good ability of degrading the extracellular matrix and penetration into tumor tissues, which improved the immunotherapeutic efficacy of solid tumors. Conclusion This paper successfully introduced HAase on the surface of therapeutic immune cells through a fucosyltransferase-based method, which increased infiltration of immune cells into the tumour site and thus enhanced the penetration into solid tumors, and provided new references for the treatment of solid tumors, as well as new protocols for exploring the new generation of NK cells for tumor immunotherapy. |
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