基于物理过滤与原位杂交技术的骨肉瘤循环肿瘤细胞检测及临床意义  

Detection and Clinical Significance of Circulating Tumor Cells in Osteosarcoma Patients using Physical Filtration combined with in Situ Hybridization

作　　者：李明辉[1] 鲁亚杰[1] 张浩强[2] 高鹏[1] 杨迪 陈国景[1] 王臻[1] LI Ming-hui;LU Ya-jie;ZHANG Hao-qiang;GAO Peng;YANG Di;CHEN Guo-jing;WANG Zhen(Department of orthopedic,Xi-Jing Hospital,The Fourth Military Medical University Xi＇an,Shaanxi,710032,China;Department of Joint Surgery,General Hospital of Lanzhou Military Region,Gansu,Lanzhou,730050,China)

机构地区：[1]第四军医大学西京医院骨科,陕西西安710032 [2]兰州军区兰州总医院骨科中心,甘肃兰州730050

出　　处：《现代生物医学进展》2018年第12期2238-2241,2293,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金面上项目(31370944;31170914)

摘　　要：目的:探寻一种有效地从骨肉瘤患者外周血中富集并鉴定循环肿瘤细胞的方法。方法:利用基于物理过滤与原位杂交结合的技术对骨肉瘤患者外周血循环肿瘤细胞分离并鉴定。采用直径8μm纳米滤膜截留外周血中体积较大的白细胞及肿瘤细胞,利用多重RNA原位杂交技术检测CD45、EpCAM、CK8、CK18、CK19、vimentin及twist基因表达,并根据结果对滤膜截留下的细胞进行鉴定并分型。结果:本研究所使用的基于物理过滤与原位杂交技术的循环肿瘤细胞检测方法可以高效地从骨肉瘤患者外周血中富集骨肉瘤循环肿瘤细胞,该方法富集细胞的效率超过90%。15例健康志愿者中1例志愿者检测结果阳性。20例纳入研究的骨肉瘤患者中19例患者外周血中检测出CTC,CTC计数范围为0-20。肿瘤转移患者外周血CTC计数为11.33±5.88,肿瘤未转移患者外周血CTC计数为4.36±2.98,差异具有统计学意义(P=0.0022)。肿瘤转移患者外周血间质型CTC比例高于肿瘤未转移患者(P=0.0031)。结论:利用基于物理过滤与原位杂交结合的技术可以有效地检测骨肉瘤患者外周血循环肿瘤细胞。CTC检测结果可以作为辅助判断肿瘤转移情况的辅助指标。Objective： To investigate the detection and clinical significance of circulating tumor cells in osteosarcoma patients using physical filtration combined with in situ hybridization. Methods： In this study, the detection of circulating tumor cells in osteosarcoma is based on physical filtration combined with in situ hybridization. The cells are firstly enriched from peripheral blood by 8 μm diameter pores calibrated membrane. Then multiplex RNA-in situ hybridization assay was used to characterize the molecular markers including CD45, Ep CAM, CK8, CK19, Vimentin and Twist in each cell. The cells are classified as leukocyte and circulating tumor cell base on the expression of different markers. 20 osteosarcoma patients and 15 healthy donors were enrolled to investigate the clinical significance of circulating tumor cells. Results： The cell recovery rate was higher than 90%. Among the enrolled 20 patients and 15 healthy donors, 19 patients and 14 healthy donors had been detected CTCs in peripheral blood. CTC count in metastatic and localized patients is 11.33 ±5.88 and 4.36±2.98, respectively. The result revealed that CTC count in metastatic patients is significantly higher than localized patients.The ratio of mesenchymal CTC in metastatic patients is significantly higher than localized patients. Conclusions： Circulating tumor cells in osteosarcoma can be detected efficiently by physical filtration combined with in situ hybridization. CTC count and phenotype are related with tumor metastasis in osteosarcoma.

关 键 词：循环肿瘤细胞 骨肉瘤 液体活检 肿瘤转移 

分 类 号：R-33[医药卫生] R738