多发性骨髓瘤中人类疱疹病毒8型的定量分析及其相关基因的表达

Quantitative analysis of human herpes virus type 8 and expression of its genes in patients with multiple myeloma

目的 确定多发性骨髓瘤 (MM)骨髓活检标本中是否存在人类疱疹病毒 8型 (HHV8)感染 ,病毒负荷量的差异与临床表现之间的关系。方法 实时荧光定量PCR确定骨髓活检标本中的HHV8病毒的负荷量 ,采用筑巢式PCR扩增患者外周血、骨髓穿刺和骨髓活检标本中HHV8KS330 2 3 3Bam片段 ,用逆转录 PCR方法了解HHV8病毒的致病基因病毒源白细胞介素 6 (vIL 6 )和病毒源干扰素调节因子 1(vIRF 1)的表达情况。结果 多数MM患者的骨髓活检标本可检出HHV8,荧光定量PCR发现阳性率为 6 9 6 % (16 / 2 3) ;筑巢式PCR时的阳性率为 82 6 % (19/ 2 3) ,骨髓穿刺和外周血标本的阳性率分别为 4% (1/ 2 5 )和 0。临床分析表明病毒的检出可能与化疗有关。vIRF 1在骨髓活检标本中表达率很高 ,为 83 3% (10 / 12 ) ,vIL 6的表达率为 0。结论 多发性骨髓瘤和HHV8感染有关 ,高表达的vIRF

Objective To investigate if infection of human herpesvirus type 8 (HHV8) exists in Chinese patients with multiple myeloma (MM), and to identify the relationship between clinical manifestation and the viral load in bone marrow biopsy samples. Methods Real time quantitative PCR was conducted to define the viral load of HHV8 in bone marrow biopsy samples from 23 patients with MM. Nested PCR was conducted to amplify the KS330 233 Bam fragment of HHV8 from peripheral blood mononuclear cells, bone marrow aspirates and bone marrow biopsy samples. RT PCR was used to study the expression of suspected oncogenes: vIL 6 and vIRF 1 genes. Bone marrow biopsy samples of 5 patients with blood diseases other than MM and 5 patients with non blood system diseases were used as controls. PCR product of BCBL 1 cell line was used as positive control. Results (1) The positive rate of HHV8 in bone marrow biopsy samples from patients with MM was 69 6%(16/23)by real time fluorescence quantitative PCR, and 82 6%(19/23)by nested PCR. 16 bone marrow samples positive by fluorescence quantitative were positive too by nested PCR. However, 3 bone marrow samples positive by nested PCR were negative by fluorescence quantative. HHV8 was positive in only one of the 25 bone marrow aspirates from patients with MM (4%) and in none of the 20 peripharal blood samples from patients with MM. PCR showed negative result in 7 bone marrow aspirates taken form 13 MM patients with positive result by fluorescence quantative PCR. HHV8 was negative in bone marrow biopsy samples from patients with other diseases. HHV8 was negative in the peripheral blood samples from 16 MM patients, among which 9 out of the 11 cases with bone marrow biopsy samples taken showed HHV8 positive by fluorescence quatative PCR. Bone marrow biopsy samples of all patients with other blood diseases and non blood system diseases were HHV8 negative. RT PCR showed that among 12 MM patients the expression rate of vIRF 1 was 83.3%(10/12), and expression rate of vIL 6 was 0. The PCR positive products tallied with the sequence of HHV8 DNA. Clinical analysis showed that all of the patients with negative results had had chemotherapy. Conclusion There is a certain association between multiple myeloma pathogenesis and HHV8. In detection of HHVb, nested PCR is more sensitive than fluorescence quantitaative PCR. The viral gene vILR 1 plays a certain role in the pathogenesis of MM.