骨肉瘤常发生肺微转移造成预后差，临床缺乏有效的预警技术。外泌体由肿瘤细胞产生、释放并调控转移，血液中外泌体含量敏感地反映肿瘤转移程度；RANKL指示着骨肉瘤的发生、发展，与骨肉瘤外泌体联合检测，能有效监控骨肉瘤微转移。在前期研究基础上，利用骨肉瘤外泌体膜表面表达CD81、MHC-I、整合素α6β4和α6β1，结合磁珠分离技术，建立免疫夹心法识别反映骨肉瘤微转移的外泌体。通过生物素-亲和素放大系统引入Catalase催化调节H2O2与HAuCl4之间的反应，利用产生对应不同浓度目标物的纳米金可视化信号，准确、重现灵敏地检测骨肉瘤外泌体。再结合免疫夹心法可视化高灵敏准确检测RANKL，建立血液外泌体和RANKL联检新平台，测定骨肉瘤临床样本，并与良性病变组和健康者的测定结果对照，构建基于外泌体和RANKL联合检测值的数据逻辑分析方法诊断预警骨肉瘤微转移，指导临床制定合理的个体化治疗方案。

The prognosis of patients with osteosarcoma is poor due to the common micrometastases of osteosarcoma towards lung, and there is a lack of effective early warning technology. The exosomes produced and released by tumor cessl can regulate the metastasis of tumor. The content of exosomes in blood can sensitively reflect the progress degree of tumor metastasis. RANKL indicates the occurrence and development of osteosarcoma, the combined detection of RANKL and the exosomes from osteosarcoma can effectively monitor the micrometastases of osteosarcoma。On the basis of previous research, we propose that combined the expression CD81,MHC-I, integrin α6β and α6β1 on the membrane of exosomes of osteosarcoma and the magnetic separation technology, an immune sandwich method for the identification and testing of the exosomes of the micrometastasis of osteosarcom can be fabricated. The introduction of Catalase through biotin-avidin amplification system can modulate the reaction between H2O2 and HAuCl4, the different visual signals from the fabrication of Au nanoparticles can be applied to quantify the concentrations of targets. Thus, the concentration of exosomes of osteosarcoma will be tested accurately, reproducibly and sensitively. Combined the sensitive and accurate sensing RANKL by colormetric signals from Au nanoparticles, the combined detection of blood RANKL and exosomes will be constructed. Detecting the clinical samples of osteosarcoma, comparing with the results of those samples of benign lesion group and healthy people, statistical data and logic methods will be fabricated according to the testing results of exosomes and RANKL. Furthermore, the fabricated methods can guide the clinical development of rational individualized treatment plan for osteosarcoma.

本项目在国家自然科学基金的资助下，主要开展骨肉瘤细胞培养，外泌体提取、蛋白验证以明确骨肉瘤细胞外泌体的结构表达；可作为目标物调节而改变的荧光信号指示体系的研究等。建立以差速离心法和试剂盒提取方法为代表的行之有效的外泌体提取方法；TEM和WB验证所获取骨肉瘤细胞外泌体的特征囊泡状结构和CD63、TSG101表达；设计并建立一种因目标调整而引起的酪氨酸酶浓度或者pH变化导致的信号强度改变的敏感石墨烯量子点调节体系，为建立三明治夹心法型的免疫分析体系建立信号基础。本项目的进行为后续研究，比如外泌体表面特征指示骨肉瘤肺转移蛋白性质的确定、以外泌体为载体的骨肉瘤微转移动物模型的建立和三明治夹心法测定外泌体等建立外泌体提取、表征方法和信号指示体系奠定了基础。一篇SCI论文在投，培养硕士研究生2名。本项目的后续深入研究仍在进行。