实时定量PCR检测多发性骨髓瘤骨髓细胞黑色素瘤抗原-C1/癌睾丸抗原7基因的表达水平

Expression level of MAGE-C1/CT7 gene in bone marrow cells with multiple myeloma by real-time quantitative PCR

目的研究多发性骨髓瘤(MM)患者骨髓单个核细胞中黑色素瘤抗原-C1/癌睾丸抗原7(MAGE-C1/CT7)基因的表达水平并初步探讨其临床意义。方法制备含MAGE-C1/CT7基因克隆片段的质粒标准品,采用实时定量PCR技术制作定量评估MAGE-C1/CT7拷贝数的标准曲线,并利用该技术检测MM患者骨髓细胞中MAGE-C1/CT7基因的表达水平。来自同种基因移植供者的正常人骨髓细胞作为对照。结果检测含MAGE-C1/CT7基因克隆片段的质粒的灵敏度可达1个拷贝,在MAGE-C1/CT7表达阳性的患者样本中,其检测灵敏度可达10-5～10-4。在77份MM患者骨髓样本中,有66份检出MAGE-C1/CT7基因表达,阳性率达85.7%,其中初治、难治复发患者中表达阳性率达88.3%(45/51),同时检测的22例正常供者骨髓样本均为阴性。此外,MAGE-C1/CT7表达量与骨髓浆细胞数呈正相关(r=0.435,P<0.05,n=25),与流式细胞术检测的CD38+/CD138+细胞数呈正相关(r=0.410,P<0.05,n=31)。结论建立的实时定量PCR可灵敏地检测MM患者MAGE-C1/CT7基因表达水平,而MAGE-C1/CT7基因水平可能成为临床辅助诊断MM的一个参考指标。

Objective To measure the expression level of MAGE-C1/CT7 gene in multiple myeloma and to explore initially its clinical implication.Methods The plasmids containing cloned fragments of MAGE-C1/CT7 gene were prepared.Standard curves from the plasmids were made by the real-time quantitative polymerase chain reaction(RQ-PCR) assay.The RQ-PCR assay was used to quantify the MAGE-C1/CT7 mRNA copy numbers in the bone marrow mononuclear cells from patients with multiple myeloma.Normal marrow samples from the allogeneic stem cell transplantation donors were served as control.Results The detection sensitivity in plasmids containing cloned fragments of MAGE-C1/CT7 gene was 1 copy,and the detection sensitivity in positively expressing MAGE-C1/CT7 gene from the bone marrow specimens was approximately in the range of 10-4 ~10-5.The positive rate with expression of MAGE-C1/CT7 gene was 85.7% in 77 multiple myeloma patients(66/77),and that was 88.3% in 51 newly diagnosed and relapsed patients(45/51).The expression of MAGE-C1/CT7 gene was not detected in the bone marrow specimens from 22 normal volunteers.A significant correlation was obtained between the expression levels of MAGE-C1/CT7 gene and the percentages of plasma cells in bone marrows(r=0.435,P<0.05,n=25).In addition,an evident correlation was obtained between the expression levels of MAGE-C1/CT7 gene and the percentages of CD38 positive/CD138 positive cells in bone marrow as well(r=0.41,P<0.05,n=31).Conclusions The established RQ-PCR method is able to sensitively measure the expression level of MAGE-C1/CT7 gene in patients with multiple myeloma,and the expression levels of MAGE-C1/CT7 gene may be a useful clinical indicator for auxiliary diagnosing multiple myeloma.