A Study On The Expression Of CD131 An Its Role In Acute Myeloid Leukemic Cells

Objects:1. To explore whether the expression of CD131 is abnormal in acute myeloid leukemic cells, the expression of CD131 was detected in peripheral blood mononuclear cells from acute myeliod leukemia patients and healthy donors, 2. To explore the relationship between the function of wild type CD131 and the biological behaviour of NB4 cells , wild type CD131 was transfered into NB4 cells via lentivirus carrying wild type CD131 to make NB4 cells over-exprssed wild type CD131,named NB4-CD131 cells, and the changes of proliferation and differentiation behaviour were detected in NB4-CD131 cells with the stimulation of IL-3 or GM-CSF.Methods: 1.The peripheral blood from acute myeliod leukemia patients and healthy dornors were collected, and the expression level of CD131 mRNA was detected in acute myeloid leukemia and heathy donors by RT-PCR. The full length coding sequences of CD131 from patients and healthy donors was amplified by RT-PCR,and linked into pGEM-T clone vector, and then the vector was transformed into Top10 competent cells, 8 positive recombinant clones of each sample was picked up and the full length coding sequences were analysed by DNA sequecing, 2. The ORF of wide type CD131 was amplified by PCR from the the cDNA of healthy donors and cloned into lentivirus vector pRRLSIN.cPPT.PGK/IRES/GFP.WPRE to construct a lentiviral vector carrying the ORF of CD131 named pLV-CD131. And the pLV-CD131 plasmid was confirmed by digesting and sequencing. The recombinant lentivirus were produced by co-transfected three plasmids into 293T packing cells. After the lentiviral supernatant infected NB4 cells, GFP expression was observed by fluorescent microscope and the expression of CD131 was detected by Western blot, 3. 4 groups of NB4 cells over-expressed of wide type CD131,named NB4-CD131, were prepared and treated with 10ng/ml GM-CSF,10ng/ml IL-3,1μM ATRA respectively. NB4 cells infected by blank lentivirus, named NB4 Blank, were treated with the same method as negatived control. The proliferation and differentiation of every group were detected by cytometry,flow cytometry,and wright staining respectively.Results:1.RT-PCR shows that the average of relative gray scale was 0.187± 0.130 in 44 cases of acute myeloid leukemia patients,and that was 0.438±0.127 in 24 health donor group(P<0.001). The mutation rate of 141 clones from acute myeloid leukemia was 73.8% and that was 20.4% in 49 clones from healthy dornor. 2. NB4 cells were stablely over-expressed wide type CD131 by transfered with recombinant lentiviral vector carrying CD131. The expression of CD11b was up-regulated in NB4-CD131 cells with the stimulation of IL-3 or GM-CSF, but lower than that in NB4-CD131 cells treated with ATRA, and morphology change was not observed in NB4-CD131 cells treated with IL-3 or GM-CSF. IL-3 or GM-CSF also didn't effect the NB4-CD131 cell proliferation.Conclusions: CD131 was expressed at a low level and high frequency of mutation in some acute myeloid leukemic cells. IL-3 or GM-CSF can induce NB4 cells over-expressed of wide type CD131 differentiation at a limited degree but had nothing effect on NB4-CD131 cells proliferation.