骨肉瘤细胞外泌体生物信息谱的建立及其对骨肉瘤患者术后复发和转移的预测研究

Osteosarcoma is the most often sarcoma for the bone, which is predilection for teenagers and cause more harm to the countrymen. Although radiological assessment is the important approach for the monitoring the recurrence and metastasis after the normalized treatment, diagnostic delay often occurs due to identifying it after the recurrence and metastasis. The radiological assessment lacks the ability of prewarning of the adverse prognosis. In this project, based on the understanding of the effect of inducing the tumor metastasis and from the perspective of the cells in vitro, we will apply exosome isolation technology and high throughput detection technology of protein and gene to establish the biological information spectrum. We apply the molecular biological technique to assess the interesting protein and gene. Combining with the prognosis of data base and the exosome of the primary culture cell, the biological information spectrum can prewarn the recurrence and metastasis. Meanwhile, based on the biological information spectrum, this project will establish the super sorting method whose the molecular target is the specific marker. This project is aiming to monitor the prognosis for the osteosarcoma patient, which is expected to provide a new technological which is easy operating, sensitive and accurate detection.

骨肉瘤是最常见的骨的原发性恶性肿瘤，好发于青少年，严重危害国民身体健康。目前临床中所采用的影像学手段是骨肉瘤患者规范化治疗后复发和转移监测的重要手段，但其多在复发和脏器转移后才可发现，存在一定的滞后性，缺乏对患者不良预后的超前预警能力。基于肿瘤源性外泌体可以促进肿瘤细胞远隔转移的认识，本项目拟从体外细胞水平为切入点，以多株骨肉瘤细胞系为平台，利用外泌体分离技术、蛋白和基因组学高通量检测技术建立骨肉瘤细胞系外泌体生物信息谱，并采用分子生物学技术对筛选获得的兴趣蛋白和基因再次验证，同时联合骨肉瘤患者肿瘤标本原代培养细胞和血液中的外泌体蛋白和基因表达特点及随访数据库信息，揭示骨肉瘤外泌体生物信息谱表达特点对骨肉瘤患者复发和转移的超前预警作用。通过本项研究以期实现通过检测筛选出的骨肉瘤患者血液中外泌体兴趣蛋白和基因的表达情况，为骨肉瘤患者预后监测提供一种操作简便、检测灵敏和准确性高的预测手段。

本项目通过比较骨肉瘤患者血清外泌体全转录组同健康人群之间的差异，建立骨肉瘤患者血清外泌体生物信息谱。在全转录组差异分析结果基础上，揭示骨肉瘤源性外泌体可能参与骨肉瘤侵袭迁移和免疫逃逸等过程。通过提取患者血清源性外泌体和骨肉瘤细胞悬液外泌体后，在体外细胞水平验证骨肉瘤患者血清外泌体和骨肉瘤细胞悬液外泌体可显著促进骨肉瘤细胞的侵袭迁移过程。利用基因修饰技术改造骨肉瘤细胞Rab27a表达水平从而影响细胞外泌体的分泌能力，借助不同外泌体分泌能力的骨肉瘤143B细胞构建骨肉瘤肺转移模型，观察肺部病灶进展情况，结果显示，Rab27a敲减细胞的143B细胞肺转移能力明显减弱，说明通过抑制骨肉瘤外泌体分泌能力可以实现抑制骨肉瘤肺部病变的进展。通过Western blot和酶联免疫吸附技术比较骨肉瘤患者、良性骨肿瘤患者和健康志愿者之间血清外泌体PD-L1和N-cadherin的表达水平，结果显示骨肉瘤患者血清外泌体PD-L1和N-cadherin表达水平显著高于另外两者，同时通过比较骨肉瘤肺转移患者和无肺转移患者血清外泌体中PD-L1表达水平，结果显示骨肉瘤患者血清外泌体中PD-L1明显高于无肺转移者。以上实验结果证实，骨肉瘤外泌体参与疾病的进展过程，通过监测骨肉瘤患者血清外泌体中特定蛋白表达水平的变化情况，可以良好预测骨肉瘤患者肺转移事件的发生。