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Detection of acute myeloid leukemia cells CD131 expression, understanding the purpose of: 1. Acute myeloid leukemia cells CD131 expression with or without abnormal; 2. NB4 cells to target cells with lentivirus-mediated gene transfer method, NB4 cells over expression The wild-type CD131, observed over-expression of wild-type CD131 NB4 cells in IL-3, GM-CSF under proliferation and differentiation of behavior change and to explore the relationship between CD131 and leukemia cell biology behavior. Method: 1. Collected 44 cases of acute myeloid leukemia primary treatment of peripheral blood mononuclear cells, 24 cases of normal human peripheral blood mononuclear cells as a control, RT-PCR CD131 in the peripheral blood of acute myeloid leukemia patients and normal controls mononuclear cell expression levels, while selected 15 patients and 5 normal PCR amplification CD131 full-length coding sequence, connected to the pGEM-T vector transformed into Top10 competent cells, positive clones were screened, sequence analysis detection of acute myeloid mutations in the coding sequence of the leukemia cells CD131. Normal human peripheral blood mononuclear cell cDNA as a template, using primer PCR amplification of the CD131 coding sequence with PmeI and BstBI restriction sites, digested cloned into the lentiviral vector pRRLSIN.cPPT.PGK / IRES / GFP . WPRE, build a lentiviral vector containing wild-type CD131 gene by restriction enzyme digestion and sequencing to verify its correctness, will build a good vector plasmid with the packaging plasmids were co-transfected 293T cells expressing wild-collected culture supernatant, low infection type CD131 in NB4 cells, Western Blot identification of target gene expression. The over-expression of wild-type CD131 cells (NB4-CD131 cells) were divided into four groups: were added to all-trans retinoic acid (final concentration of 1 μM), GM-CSF (final concentration of 10ng/ml), IL-3 (final concentration of 10ng/ml) and the volume of culture. NB4 cells transfected with empty vector (NB4-Blank) as a negative control. Comparison groups cell proliferation, cell counting and flow cytometry analysis and comparison of each group of CD11b expression Wright stain under a microscope, observed changes in cell morphology. Results: 1.RT-PCR 44 patients with acute myeloid leukemia cases and 24 cases of normal human peripheral blood mononuclear cells CD131 average relative gray value were 0.187 ± 0.130 and 0.438 ± 0.127, AML group CD131 expression levels significantly lower than normal control group (P = lt; 0.001); 141 from AML cases CD131 full-length coding sequence, 104 mutation, the mutation rate was 73.8%, 49 the CD131 coding sequences from normal mutation rate of 20.4%, the AML group the CD131 mutation rate significantly higher than the normal control group (P = lt; 0.001); the recombinant CD131 gene lentiviral effective infection NB4 cells, and NB4 cells overexpression CD131. Over-expression of CD131 in NB4 cells, GM-CSF, IL-3, RA role, CD11b upregulation RA group, but the magnitude of increase obvious the NB4-CD131 cells in the GM-CSF or IL-3 morphological changes were not observed. Simultaneously overexpressed CD131 in NB4 cell proliferation without change, no change in IL-3 or GM-CSF stimulated proliferation. Conclusion: 1.CD131, in acute myeloid leukemia cells express low levels and mutation rates; 2, IL-3, GM-CSF to a certain extent induced over-expression of wild-type CD131 in NB4 cell differentiation to neutrophils early , but the proliferation of no effect, also did not justify over-expression of wild-type CD131 NB4 cells fully differentiated mature.
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