Expression of CD131 Gene in Acute Myeloid Leukemic Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To detect the expression level and the mutantion of CD131 in acute myeloid leukemic (AML) cells, and to analyze the relationship of CD131 expression level with clinical features.</p><p><b>METHODS</b>The peripheral blood mononuclear cells (PBMNC) from 44 AML patients and 25 healthy donors were collected, and the expression level of CD131 mRNA was detected by RT-PCR. The full length coding sequence of CD131 from 15 patients and 5 healthy donors was amplified by RT-PCR, and linked to pGEM-T vector to clone TA, 10 positive recombinant clones of each sample were analyzed by DNA sequencing. The AML patients were divided into CD131 negative and CD131 positive groups according the expression level of CD131, and white blood cell counts, CD34(+) cells percentage, complete remission rate of patients were compared.</p><p><b>RESULTS</b>CD131 was expressed at a lower level in AML than that in healthy donor. Five kinds of CD131 mutantions could be detected in both AML and healthy donor groups, but the mutation rate in AML (75.33%) was higher than that in healthy donors (18.0%). CD131 negative group showed a higher CD34(+) cells percentage (69.1% ± 20.8%), and lower complete remission rate (33.3%) than that in CD131 positive group (69.1% ± 20.8%, 33.3%). No statistically significant difference of WBC counts was found between 2 groups.</p><p><b>CONCLUSION</b>CD131 is expressed at a low level and shows high frequency of mutation in acute myeloid leukemic cells. CD131 negative AML correlats with high proportion of CD34(+) cells, and showed insensitive to chemotherapy.</p>

Packaging of lentivirus carrying gene hβc and overexpression of gene hβc in NB4 cells / 中国实验血液学杂志

This study was aimed to overexpress gene hβc in NB4 cells via the method of lentivirus-mediated gene transfer, to observe the differentiation behaviour change of hβc over-expressing NB4 cells treated with IL-3 or GM-CSF, to explore the relationship between hβc gene and the differentiation behaviour of NB4 cells. The targeted hβc gene was amplified by PCR from the cloned vector carrying ORF of hβc. The PCR product containing PmeI and BstBI site introduced by primer was digested, and then cloned into lentivirus vector pRRLSIN.cPPT.PGK/IRES/GFP.WPRE to construct a lentiviral vector carrying hβc, named pLV-hβc. And the pLV-hβc plasmid was confirmed by restriction and sequencing. The recombinant lentivirus was produced by co-transfecting three plasmids into 293T packing cells. After transfection, the lentiviral supernatant was collected to transfect NB4 cells. GFP expression was examined by fluorescent microscope and the expression of hβc gene was detected by Western blot. Then, the NB4 cells over-expressing hβc were treated with IL-3 (10 ng/ml), GM-CSF (10 ng/ml), ATRA (1 µmol/L) respectively, and the CD11b expression, morphology and differentiation behaviour changes of every groups were observed by flow cytometry and microscopy, while NB4 cells transfected with blank lentivirus (NB4-blank cells) were used as controls. The results showed that the recombinant lentivirus vector carrying hβc gene could efficiently transfect NB4 cells and made NB4 cells to stably over-express hβc gene. The expression of CD11b was up-regulated in NB4-hβc cells treated with of IL-3 or GM-CSF, but it was not as obvious as the effect of ATRA, and no morphological change was observed in NB4 hβc cells treated with the IL-3 or GM-CSF. It is concluded that IL-3 or GM-CSF can induce NB4 cells over-expressing hβc to differentiate to neutrophils, but can not make them fully matured.

Expression change of IL-3 receptor system in all-trans retinoic acid induced differentiation of NB4 cells / 中国实验血液学杂志

Interleukin-3 receptor (IL-3R) is a heterodimeric membrane receptor. The α subunit is essential for ligand binding and confers ligand specificity to the receptor. The common beta chain (βc) subunit, which is shared by the granulocyte macrophage-colony stimulating factor (GM-CSF), IL-3 and IL-5 receptors, is required for high-affinity ligand binding and signal transduction, mediating growth and survival of hematopoietic progenitor cells and the production and activation of mature hematopoietic cells. In order to investigate the role of IL-3 receptor system (IL-3Rα, GM-CSFRα and hβc) in myeloid differentiation, the expression level of IL-3 receptor system gene in all-trans retinoic acid (ATRA)-induced NB4 cell differentiation was detected by quantitative real time RT-PCR. At the same time, DNA sequence change was analyzed by cDNA sequencing. The results showed that the expression level of IL-3Rα mRNA was obviously down-regulated in NB4 cells treated with ATRA for 24 hours, but during differentiation of ATRA induced NB4 cells, the expression level of IL-3Rα mRNA was gradually restored, while the expression levels of GM-CSFRα mRNA and hβc mRNA were gradually up-regulated. The sequence of IL-3Rα and GM-CSFRα gene did not change before and after NB4 cells differentiation, but the sequence of hβc gene changed when NB4 cells were treated with ATRA, the expression of hβc mRNA sequence before NB4 cell differentiation taken truncated mutation as dominant, as regards expression of hβc mRNA sequence after NB4 cell differentiation, the truncated mutation of hβc mRNA had restored to wild type. It is concluded that the IL-3 receptor abnormality exists in NB4 cells, over expression of IL-3Rα and truncated mutation of hβc may be involved in proliferation and differentiation block in NB4 cells.