| Objective1. To examine human herpesvirus 8 (HHV-8) DNA in peripheral mononuclear cells (PBMCs) from blood donors of Shenyang, China, and to analyze whether there were differences among different sex, age, ethnic groups and blood types.2.To examine HHV-8 DNA in PBMCs, bone marrow mononuclear cells (BMMCs), bone marrow biopsy samples and biopsy samples of lymph node and lymphomatous tissue from patients with lymphoma, leukemia and multiple myeloma, to investigate the possible role of HHV-8 in the pathogenesis of these diseases. And detect T lymphocyte subsets simultaneously to investigate relation between HHV-8 and patient's immune state.3.To examine IL-6 level in sera from patients with multiple myeloma, and analyze the association of HHV-8 with IL-6.Material and Methods1. Sample collection(l)Blood donors and normal controls110 samples of peripheral blood from healthy blood donors were collected, from which 110 samples of PBMCs, 30 samples of anticoagulant blood and sera were isolated and separated. 20 lymph node biopsy samples of reactive proliferation were collected. 15 samples of bone marrow aspirats from patients without primary hematologic diseases were isolated into BMMCs. 15 Bone marrow biopsy samples of normal rib were collected from excised tissue of patients who underwent surgical operation.(2)Patients with lymphoma36 samples of peripheral blood, 12 samples of BMMCs, 29 biopsy samples of lymphomatous tissue were obtained from 44 patients with lymphoma.(3)Patients with leukemia54 samples of peripheral blood and 32 samples of BMMCs were collected from 68 patients with acute leukemia. 6 samples of peripheral blood and 9 samples of BMMCs were collected from 13 patients with chronic leukemia.(4)Patients with multiple myeloma20 samples of peripheral bloods, 24 samples of BMMCs and 16 bone marrow biopsy samples were collected, and from peripheral blood 20 samples of serum were separated.2. DNA extraction and concentration measurement and adjustment (l)PBMCs and BMMCs were isolated from peripheral blood and bonemarrow aspirats samples with Ficoll-Hypaque.(2)DNA was extracted from PBMCs of blood donors, and from PBMCs, BMMCs, bone marrow biopsy samples and biopsy samples of lymphomatous tissue from patients with lymphoma, leukemia and multiple myeloma with standard phenol-chlorophorm method.(3)DNA concentration and purity were measured with spectrophotometry at 260nrn and 280nm, the concentration was adjusted to 1g/l with TE buffer.3. Primer designPrimer set KS1/KS2 designated as HHV-8 KS330 primer, was designed to amplify the highly specific region of HHV-8 KS330 Bam gene fragment.4. PCR amplificationPCR with KS330 primer KS1/KS2 was undertaken to detect the KS330 Bam fragment of HHV-8 in PBMCs from blood donors, and in PBMCs, BMMCs, bone marrow biopsy samples and biopsy samples of lymphomatous tissue from patients with lymphoma, leukemia and multiple myeloma.5. Recovery of PCR amplified productsThe amplified products of 233bp by KS1/KS2 were retrieved with DNA fragment recovery kit.6. Restricted endoneuclease analysis of the PCR productsThe 233bp products amplified by primer set KS1/KS2 were cleaved by restricted endoneuclease Pstl. Then the cleaved products were identified withagrose gel eletrophoresis or polyacrylamide gel electrophoresis (PAGE).7. T lymphocyte subsets detection with flow cytometryT lymphocyte subsets of peripheral blood from blood donors and patients with lymphoma, leukemia and multiple myeloma were detected with murine-anti-human CD3-PE, CD4-FITC, CD8-FITC monoclonal antibodies and flow cytometry.8. Detection of IL-6 level in sera by ELISAThe concentration of IL-6 in sera from blood donors and patients with multiple myeloma were examined with standard ABC ELISA method with human IL-6 quantitative determination ELA kit.9. Statistical analysis(l)Chi-square (x2) test was used to compare the detection rates of HHV-8 DNA among blood donors of different sex, age, ethnic groups and blood...
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