Expression of miR-9 in B lymphocytes and B cell lymphomas cell lines and its significance / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of miR-9 in B lymphocytes, B cell lymphoma and classical Hodgkin's lymphoma (cHL) cell lines and its significance.</p><p><b>METHODS</b>CD19(+) B lymphocytes were sorted from normal lymph node by magnetic beads. Total cellular micro-RNA was extracted from cHL cell line L428, B cell lymphoma cell lines Ly1 and Ly10 (diffuse large B cell lymphoma), Raji cells (Burkitt's lymphoma) and CD19(+) B lymphocytes, respectively. These micro-RNAs were separately transformed into cDNA by reverse transcription. The expression levels of miR-9 were measured by fluorescence quantitative PCR. In situ hybridization was used to detect the expression of miR-9 in cell lines.</p><p><b>RESULTS</b>The expression of miR-9 was high in L428 cells (104.44 ± 1.61), and low in cell lines of B cell lymphoma (Ly1: 2.17 ± 0.38; Ly10: 1 ± 0.015; Raji: 2.65 ± 0.89), and extremely low in CD19(+) B lymphocytes (0.0026 ± 0.00040). Compared with that in the other cell lines, the expression of miR-9 in L428 cells was statistically significant (P < 0.05). miR-9 localized in the cytoplasm diffusely and strongly in L428, but scattered and slightly with some prominent distribution around the nuclear membranes in Ly1 and Ly10, and only weakly in Raji.</p><p><b>CONCLUSIONS</b>miR-9 highly expressed in cHL cell line and might be a molecular marker for diagnosis and treatment of cHL.</p>

Preparation of RNA probe for cd99l2 gene of zebrafish labeled with digoxingenin-UTP / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the expression pattern of cd99l2 gene during zebrafish development, the RNA probes for whole-mount in situ hybridization were prepared in this study.</p><p><b>METHODS</b>The cd99l2 fragment obtained by RT-PCR was cloned into pGM-T Easy, then the plasmids were linearized with the restriction enzymes SacII or SalI. Using Sp6 or T(7) RNA polymerase, the digoxingenin-labeled antisense and sense probes were synthesized and confirmed by whole-mount in situ hybridization.</p><p><b>RESULTS</b>The plasmid cd99l2/pGM-T was constructed. cd99l2 gene expression pattern during embryogenesis of zebrafish was examined using the antisense probe, and intense expression was detected in the central nervous system during zebrafish development.</p><p><b>CONCLUSION</b>The antisense probe can be used for study of the spatial and temporal distribution of cd99l2 during zebrafish development using the sense probe as control.</p>

Primers for detecting gene rearrangement in different regions of immunoglobulin heavy chain genes and their application in diagnosis of paraffin-embedded lymphoma tissues / 南方医科大学学报

<p><b>OBJECTIVE</b>To analyze and optimize the gene rearrangement primers of different frame regions (FR) of immunoglobulin heavy chain (IgH) genes by bioinformatic methods and explore the application of these primers in the detection of paraffin-embedded lymphoma tissues.</p><p><b>METHODS</b>Three pairs of primers from IgH FR1, FR2 and FR3 regions (P1c, P2A and P31, respectively) were selected as the B cell gene rearrangement primers after comparison of the gene fragments in 44 IgH variable and 6 joining regions. Using one pair of T cell receptor (TCR) gamma primer as the T cell gene rearrangement primer, 101 histopathologically confirmed lymphoproliferative samples including 80 B cell lymphomas, 14 T cell lymphomas, and 7 reactive proliferative lymph nodes were examined by PCR for gene arrangement. The DNAs from DG75 and Jurkat cell lines were used as the positive controls for B and T cell lymphoma, respectively, with those from reactive proliferative lymph nodes as the negative control.</p><p><b>RESULTS</b>The positivity rates of IgH primers (P1c, P2A and P31) in the 80 B cell lymphomas were 37.5% (30/80), 52.5% (42/80) and 70.0% (56/80), respectively, and only one of the 14 T cell lymphoma cases was positive for the primers, suggesting significant differences in the detection rates of B cell lymphomas by the 3 primers. The detection rate was increased to 83.9% by combining the results by P31 and P2A primers. No positivity was found in the proliferative reaction tissues.</p><p><b>CONCLUSION</b>Primers from IgH FR3 region genes are more sensitive than that from the FR1 and FR2 regions in the detection of gene rearrangement in paraffin-embedded lymphoma tissues. The detection rates can be increased by combining the results with the primers for IgH FR3 with that of FR2.</p>

Detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma by hemi-nested PCR / 中华血液学杂志

<p><b>OBJECTIVES</b>To explore a sensitive and specific method for detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma (DLBCL), and verify the credibility of the established method.</p><p><b>METHODS</b>bcl-2/IgH hemi-nested PCR primers were designed using the professional primer design software. Fifty-two samples of pathologically diagnosed DLBCL and 10 fresh tonsil tissues were amplified using hemi-nested touch down-PCR to detect bcl-2/IgH gene rearrangement. The PCR products were cloned and sequenced.</p><p><b>RESULTS</b>bcl-2/IgH gene rearrangement was detected in 6 of 52 DLBCL samples and 2 of 10 fresh tonsil tissues using one-way method. By using the hemi-nested PCR for the second round amplification, 5 of DLBCL were positive, but all of the fresh tonsil tissues were negative. The positive PCR products were sequenced and analyzed on the Internet, 3 of 8 cases obtained by one-way method were false positive, 5 positive cases amplified using hemi-nested PCR were all bcl-2/IgH gene rearrangement. PCR products of 3 false positive cases were homologous to BAC331191 and LLNLR-245D11 in human chromosome 19 and RP11-498P10 in chromosome 1.</p><p><b>CONCLUSION</b>There are false positive results using common primers for detecting bcl-2/IgH gene rearrangement. The mechanism may be that highly homologous sequences to human genome exist in commonly used primers. The specificity of the diagnosis could be improved by hemi-nested PCR using the combination of primers we designed and the traditional ones.</p>

Unspecified peripheral T cell lymphoma with distinct lymphoid follicules / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the morphological features and immunophenotype of unspecified peripheral T cell lymphoma with distinct lymphoid follicular growth pattern.</p><p><b>METHODS</b>Three cases of peripheral T cell lymphoma with special pathohistological features were collected. Morphologic analysis and immunohistochemical staining for CD3, CD45RO, CD43, CD20, CD79a, cyclinD1, bcl-2, CD4, CD8 and S-100 were performed. PCR was used to study TCR gamma gene rearrangements.</p><p><b>RESULTS</b>The main symptoms of all the three patients with the primary sites of cervix and lower jaw. There were intermittent fever and skin rashes in the course of the disease. Morphological study showed lymphoid follicular reactive hyperplasia, mantle zone disappear, prominent infiltration of marginal zones by medium-sized tumor cells with clear cytoplasm and significant nuclear atypia. The immunophenotypic profile confirmed that they were T cell lymphomas. TCR gamma gene rearrangements were found in all the three patients.</p><p><b>CONCLUSION</b>In some unspecified peripheral T cell lymphomas, the distinct follicular growth pattern and incomplete effacement of the lymph node architecture make it necessary to differentiate them from reactive hyperplasia, marginal zone B cell lymphoma, follicular B cell lymphoma and mantle cell lymphoma.</p>