注射型改性纤维蛋白凝胶作为基因和药物缓释共载体在骨肉瘤治疗中应用研究

Osteosarcoma is a common primary malignant bone tumor, mainly in childhood and adolescence. Surgery and chemotherapy is considered as the first-line treatment of osteosarcoma, however, the treatment effect is far from the expectation. Gene therapy is an emerging targeted therapeutic strategy for the treatment of osteosarcoma. In our previous study, we found PLK1 is overexpressed in osteosarcoma, involved in carcinogenesis, progress and prognosis of osteosarcoma therapy. So we come up with the hypothesis: silencing PLK1 reveals a potential as a promising therapeutic target for osteosarcoma therapy. In the present study, synthesis of injectable modified fibrin gel and mPEG-bP(Glu)-b-P(Phe) nanogel are served as the nanogel-hydrogel system for co-loading gene and chemotherapeutics. In vitro experiments, we will study the biocompatibility, drug release and antitumor effects of the gene and drugs co-delivery system. Furthermore, the gene and drug co-delivery system carrying multi-agents chemotherapeutics and PLKs plasmid DNA are injected into the tumor site of osteosarcoma models, so that the gene and drugs can be released in the local site. Eventually we declare the combination chemotherapy effects of drugs and gene, reduce the systemic side effects of chemotherapy and study the expression of drug-resistance genes and apoptosis related gene expression to clarify the mechanism of synergistic anti-tumor efficacy of PLKs and chemotherapeutics. What’s more, it can provide a promising target for the treatment of osteosarcoma.

骨肉瘤是一种常见儿童、青少年时期原发性恶性骨肿瘤，手术治疗、联合化疗方案为目前骨肉瘤治疗一线治疗方案，但远远达不到预期效果。以基因治疗为代表的靶向治疗方案越来越受到关注。课题组前期研究表明PLK1在骨肉瘤中呈现高表达，与肿瘤的发生、发展及预后密切相关。我们提出假说：靶向沉默PLKs基因联合化疗方案可提高骨肉瘤治疗效果。本项目通过合成新型mPEG-bP(Glu)-b-P(Phe)纳米凝胶-改性纤维蛋白载药凝胶缓释体系，系统表征该载药体系生物相容性、降解性、体外基因、药物释放速率等。体内实验通过该载药体系包载化疗药物和针对PLKs靶点shRNA注射于骨肉瘤局部形成凝胶，持续释放基因和药物，靶向作用于骨肉瘤，降低药物全身副作用，研究骨肉瘤相关耐药基因及凋亡基因的表达，分析PLKs与人骨肉瘤形成、进展及预后效果的相关性，阐明PLKs与化疗药物在骨肉瘤治疗的协同作用机制，为骨肉瘤治疗提供新思路。

课题组合成了新型可注射型PLGA-PEG-PLGA温敏水凝胶，利用该水凝胶作为多种化疗药物共载体对骨肉瘤进行局部治疗。可注射型PLGA-PEG-PLGA温敏水凝胶具有良好的生物相容性和可降解性，不会引起明显的炎症反应。.利用该可注射型PLGA-PEG-PLGA温敏水凝胶作为阿霉素、顺铂药物共载体进行局部抗骨肉瘤实验。联合阿霉素、顺铂载药水凝胶可明显缓释阿霉素和顺铂，体现出明显抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡作用。体内抗肿瘤实验中，通过将载药水凝胶对荷瘤裸鼠骨肉瘤进行瘤周注射，联合阿霉素、顺铂载药水凝胶组表现出最好的抑制肿瘤生长效果。进一步研究表明，该组骨肉瘤中细胞坏死率、凋亡率明显增高，且不会引起全身各系统副作用。.合成改性纤维蛋白凝胶-ZIF作为药物载体，将阿霉素，PDL1及CD47包载于ZIF中，后将ZIF混合入纤维蛋白凝胶中，局部进行抗肿瘤实验。Zif@PCD Gel体系可明显抑制骨肉瘤细胞增殖、诱导细胞凋亡。体内实验中，通过将载有Zif@PCD Gel体系局部注射入骨肉瘤周围，表现出明显的抑制肿瘤增长效果，肿瘤组织细胞凋亡、细胞坏死率明显增高。进一步研究骨肉瘤免疫治疗及药物联合作用机制发现，联合用药组能够有效引起CD8+细胞表达，树突状细胞成熟，CD8+记忆T细胞表达，及M1细胞表达，同时诱导Caspase3的表达，下调PCNA的表达，以此达到协同抗肿瘤作用。.水凝胶具有良好生物相容性、生物可降解性，作为药物、免疫治疗载体在骨肉瘤治疗，充分发挥联合治疗优势，具有广泛应用前景。