Investigation of osteosarocma specific marker for detection of micro-metastasis

临床上非骨肉瘤可见于骨性基质，仅靠组织学检查很难与骨肉瘤相鉴别。由于骨肉瘤的治疗策略与其他恶性肿瘤有很大的不同，因此确定骨肉瘤的特异性标记物是很重要的。部分骨肉瘤患者血清碱性磷酸酶(ALP)升高，因此ALP被认为是骨肉瘤的肿瘤标志物之一。然而，我们发现一些透明细胞肉瘤也显示出血清ALP的高表达，我们认为ALP在骨肿瘤中的特异性相对较低。最近，转录因子矮小家族的成员Osf2/Cbfa1(以下简称Osf2)被发现是成骨细胞分化的激活因子。在本研究中，我们只在骨肉瘤细胞系中表达了Osf2 mRNA，而在非骨肉瘤细胞系中没有表达，这表明Osf2 mRNA的表达与骨肉瘤细胞的成骨细胞表型有关。提示CBFA1的表达可能是骨肉瘤的特异性肿瘤标志物，血液循环中的肿瘤细胞在骨肉瘤的转移过程中起中心作用。目前还没有关于循环骨肉瘤细胞与肺转移关系的报道。我们开发了一种基于酶联免疫吸附试验的聚合酶链式反应检测系统，用于检测小鼠转移模型中的循环骨肉瘤细胞。以Osf2为目的基因。发现了Osf2基因的一个剪接变异体，其表达仅限于骨骼和骨肉瘤。肿瘤转移组小鼠外周血中Osf2基因剪接变异体的含量明显高于对照组。采用Osf2基因的聚合酶链式反应-酶联免疫吸附试验是检测外周血中循环骨肉瘤细胞的一种有潜力的方法。

Clinically non-osteosarcoma tumors can be observed with osseous matrix, and it is difficult to distinguish such tumors from osteosarcomas only by histological examination. Because the treatment strategy for osteosarcoma is quite different from other malignant neoplasms, it is important to identify a specific marker for osteosarcoma. Some of osteosarcomas reveals elevation of serum alkaline phosphatase (ALP), so ALP was considered one of the tumor marker in osteosarcomas. However, we revealed some of clear cell sarcomas also showed high expression of serum ALP, and we considered ALP has relative low specificity in bone tumors.Recently, Osf2/Cbfa1 (hereafter called Osf2), a member of the runt family of transcription factors, was identified as an activator forosteoblastic differentiation. In this study, we showed the expression of Osf2 mRNA only in osteosarcoma cell lines while not in non-osteosarcoma cell lines, suggesting that Osf2 mRNA expression is related to the osteoblastic phenotype of osteosarcoma cells. This result suggested that the expression of CBFA1 might be the specific tumor marker for osteosarcoma.Circulating tumor cells in the blood play a central role in the metastatic process. There have been no reports describing the relationship between lung metastasis and circulating osteosarcoma cells. We developed a system with a polymerase chain reaction assay based on an enzyme-linked immunosorbent assay (PCR-ELISA) to detect circulating osteosarcoma cells in a mouse metastatic model. Osf2 was used as a target gene. One splicing variant of Osf2 mRNA was identified and its expression was restricted to the bones and osteosarcomas. The amount of the splicing variant of Osf2 mRNA was significantly higher in the blood of mice with metastasis than in the blood of the control group. The PCR-ELISA using Osf2 mRNA is a potential method to detect circulating osteosarcoma cells in peripheral blood.