PDCD5蛋白逆转骨肉瘤阿霉素耐药过程中互作蛋白的筛选和功能鉴定

The osteosarcoma of bone is the most common malignant bone tumor among children and adolescents. The five year survival rates have been significant improved from 10-20% to 50-60% since the combination of the neo-adjuvant chemotherapy and surgery. The adjuvant chemotherapeutic resistance is the main reason to recurrence and death for the patients suffered osteosarcoma. But there is no effective method to solve the chemotherapeutic resistance of osteosarcoma. PDCD5 is a novel apoptosis-related gene cloned as an increased expression gene during the apoptotic process of TF-1 cells induced by cytokine withdrawal by Center for Human Disease Genomics of Peking University. We firstly reported that the Adenovirus-delivered PDCD5 could counteract the Adriamycin resistance of osteosarcoma cells through enhancing apoptosis and inhibiting Pgp. This study is the further investigation of the former work. In this research program, we intend to purify the cooperative interactive protein moleculars for PDCD5 protein by the Tandem Affinity Purification (TAP) system, which is manipulated as: constructing a fusion protein of TAP and PDCD5, tranfecting the plasmid to the Saos-2 cells whose PDCD5 is knocked off, purifying the fusion protein by affinity column. Then, the biological attribute of the purified proteins would be identified by mass spectrum. Subsequently, we would apply the GSEA (gene set enrichment analysis) to explore the interactive protein moleculars' activation on the characteristic target genes of Osteosarcoma listed in the appendix. Eventually, we would conclude from the result that different interactive protein moleculars' influence on the specific biological behavior of Osteosarcoma. Furthermore, this research program would extend the mind of the Osteosarcoma related chemotherapeutic resistance's basic scientific research and made significant foundation for the signal pathway based genetic target therapy.

骨肉瘤是儿童和青少年最常见的恶性原发骨肿瘤。新辅助化疗和外科手术虽然将五年生存率由10-20%提高到50-60%，但化疗耐药一直是患者术后复发和死亡的主要原因。针对骨肉瘤化疗耐药，目前尚且没有实质性解决方法。PDCD5是北京大学免疫学实验室发现的一种TF1细胞凋亡相关基因，我们首次报道了该基因在体外有效的抑制阿霉素耐药骨肉瘤细胞的增殖，本课题是延伸研究，拟采用串联亲和纯化的方法检测PDCD5分子在耐药骨肉瘤细胞中的蛋白相互作用，分离和鉴定出与PDCD5分子特异性发生作用的蛋白质大分子，验证并确定PDCD5分子与互作蛋白的相互作用结构域，并利用基因功能集合富集度分析方法从基因组水平分析结合蛋白大分子对PDCD5分子转录激活以及细胞整体生物学行为影响。通过对特异性互作蛋白的筛选和研究，为骨肉瘤的耐药发生的分子病因学的研究，也为建立在骨肉瘤胞内信号通路基础上的靶向治疗提供实验基础和理论依据。

骨肉瘤是儿童和青少年最常见的恶性原发骨肿瘤。新辅助化疗和外科手术虽然将五年生存率由10-20%提高到50-60%，但化疗耐药一直是患者术后复发和死亡的主要原因。针对骨肉瘤化疗耐药，目前尚且没有实质性解决方法。PDCD5是北京大学免疫学实验室发现的一种TF1细胞凋亡相关基因，我们首次报道了该基因在体外有效的抑制阿霉素耐药骨肉瘤细胞的增殖，本课题是延伸研究，拟采用串联亲和纯化的方法检测PDCD5分子在耐药骨肉瘤细胞中的蛋白相互作用，分离和鉴定出与PDCD5分子特异性发生作用的蛋白质大分子，验证并确定PDCD5分子与互作蛋白的相互作用结构域，并利用基因功能集合富集度分析方法从基因组水平分析结合蛋白大分子对PDCD5分子转录激活以及细胞整体生物学行为影响。通过对特异性互作蛋白的筛选和研究，为骨肉瘤的耐药发生的分子病因学的研究，也为建立在骨肉瘤胞内信号通路基础上的靶向治疗提供实验基础和理论依据。研究结果发现：1，PDCD5分子可以有效的逆转骨肉瘤的阿霉素耐药，2，串联亲和纯化(TAP) Saos-2-TAP-PDCD5细胞中可以和TAP-PDCD5 蛋白相结合的蛋白质，并用SDS-PAGE 进行分离，用MALDI-TOF 对其进行检测，筛选出MDFI和MSB4蛋白，3，对筛选出来的MDFI和MSB4蛋白应用GST-PULLDOWN技术进行验证，结果未证实ODCD5蛋白MDFI和MSB4蛋白进行相互作用，我们后续构建MDFI和MSB4蛋白的病毒载体，应用基因芯片检测其对骨肉瘤细胞系的影响。