| ObjectiveAcute myeloid leukemia is a common malignancy in blood Disease, which characteristiced by a clonal proliferation in immature granulocytes and not accompanied with a clear differentiation. AML can be divided into a variety of clinical subtype, part of which is still not very clear. Tryptase is a serine protease, which has a variety of biological effects and currently considered as a new enzyme in AML pathogenesis, clinical monitoring process and received extensive attention. It was widely believed to be primarily secreted by mast cells and basophils. But with the extensive deeply study in tryptase, a high expression of tryptase has been found some in AML patients of part of FAB subtype. Previous research has also confirmed that acute myeloid leukemia cell line U937 can secrete tryptase. However, the role of tryptase which secreted by leukemia cells in AML is not very clear in the development of AML.Our previous findings show that the expression of VEGF and tryptase were significantly increased in AML-M2 patients, and has a significant correlation between VEGF and tryptase. Recent studies have also found that, the counts of mast cell of tryptase-positive has a positive correlation with angiogenesis in a number of malignant tumors and inflammation. Tryptase can promote overexpression of VEGF in a variety of tumor and inflammatory cells, and may serve as indicators for judging the prognosis of patients with malignant tumors. This shows that tryptase may plays a certain role in AML angiogenesis, which may be related to VEGF. PAR-2 is the cell surface receptors of tryptase, activated by tryptase and then mediating transmembrane signal transduction. Previous studies shown that, PAR-2 could transmit intracellular information by a variety of ways. The study in recent years mainly focused on MAPK signaling pathway, and the expression of VEGF was related with extracellular signal-regulated kinase ERK and p38MAPK pathways. NF-κB is an important transcription factor, which can be used as downstream signaling molecules in ERK and p38MAPK signaling pathway. Previous studies have also shown that expression of VEGF has a close relationship with NF-κB.To investigate the role of tryptase in AML angiogenesis, we will reseach that wether tryptase could increaseVEGF expression in bone marrow stromal cells of AML, and wether the mechanism of tryptase promote AML BMCs expression VEGF is that tryptase actives PAR-2, then dependenting on MAPK (ERK and/or p38MAPK) signaling pathway and activing NF-κB, increasing the expression of VEGF.Methods1. Cell culture and identification:Bone marrow and heparin sodium mixture 5ml, lymphocyte separation medium 10ml,2000rpm, 10min, mononuclear cell were obtained, then inoculated in 25ml culture flask.15% fetal bovine serum and DMEM/F12 were used to culture cells. According to cell morphology and the express of CD14, CD34, CD45, CD13 and CD117 in cell surface, which detected by FCM, AML BMSCs was identificated.2. The expression of VEGF in AML BMSCs after incubated with tryptase: MTT was used to detect cell viability after incubated with tryptase (0～50 ng/ml). In order to select the best concentration of tryptase, different concentrations (0.5,5,10 ng /ml) of tryptase were used to effect on AML BMSCs. ELISA, immunofluorescence, real time RT-PCR and Western-blot method were used to detect the expression of VEGF.3. The expression of VEGF in AML BMSCs cultured by the supernatants of U937 cells:DMEM/F12 substratum, U937 cell culture supernatant and U937 cell culture supernatant plus tryptase antibody were used to be substratum, then AML BMSCs were cultured for 24 h and VEGF mRNA were detected by real time RT-PCR method. AML BMSCs were cultured for 48 h cultured AML BMSCs 48h, and VEGF expression were detected by ELISA method.4. The expression of PAR-2 in AML BMSCs was detected by immunocytochemistry and flow cytometry surface:AML BMSCs were fixed with Paraformaldehyde, and PBS as negative contro. Immunocytochemistry was used to detect the expression of PAR-2. AML BMSCs about 10°cells were fixed with Paraformaldehyde, and incubated with FITC labeled PAR-2 for 1 hour on ice, and washed two times with PBS and re-suspended cells with 100μl PBS,-then detected by flow cytometry.5. ERK, p38MAPK protein detected by Western blot in AML BMSCs incubated with tryptase:Total cellular protein was extracted. The sample 20μg was transfered to PVDF membrane, and closed with 5% skim milk powder. The membrane were incubated overnight with ERK mouse anti-human polyclonal antibody, phosphorylated ERK mouse anti-human polyclonal antibody, and p38MAPK goat anti-human polyclonal antibody, phosphorylated ERK mouse anti-human polyclonal antibody, then incubated with respective secondary antibody at room temperature for 2 h and were colored 5min.6. The activation of NF-κB was detected EMSA in AML BMSCs after incubating with tryptase:AML BMSCs were incubated with PBS, tryptase, tryptase plus PDTC for 24h, then total nuclear protein were extracted. EMSA method was used to detect the activation of NF-κB in AML BMSCs.7. PAR-2, ERK, p38MAPK and NF-κB-specific inhibitor respectively: FLLSY-NH2 (200μM), PD98059 (20μM), SB230580 (10μM) and PDTC (100μM) were used to treated AML BMSCs for 30 min, then trypase were used to incubate with AML BMSCs, and real time RT-PCR method were used to detect the expression of VEGF mRNA.8. PAR-2, ERK, p38MAPK and NF-κB-specific inhibitor respectively: FLLSY-NH2 (200μM), PD98059 (20μM), SB230580 (10μM) and PDTC (100μM) were used to treated AML BMSCs for 30 min, then trypase were used to incubate with AML BMSCs, and Western blot method was used to detect the protein expression of VEGF.9. Statistical analysis SPSS 13.0 software was used to analysis Statistical data The measurement data was indicated with mean±standard deviation, and one way ANOVE was used to analysi with the methon of LSD P<0.05 was statistically significant.Results1. The result of ELISA, immunofluorescence, real time RT-PCR and Western-blot showed that, after AML BMSCs incubating with tryptase, compared with the control group, VEGF expression was significantly increased (P<0.05), with a dose-dependent relationship in a certain range.2. The results of ELISA and real time RT-PCR showed that, after AML BMSCs cultured with U937 cell culture supernatant, compared with the control group, VEGF expression in AML BMSCs significantly increased (P<0.05), the increased was significantly reduced by antibody of tryptase (P<0.05).3. The result of flow cytometry and immunocytochemistry showed that, tryptase-specific receptor PAR-2 was expressed on AML BMSCs.4. The results of Western blot showed that, after AML BMSCs incubated with tryptase for 48h, compared with the control group, Phosphorylation of ERK and p38MAPK protein expression were significantly increased (P<0.05), and the increasing was significantly reduced by PD98059 and SB230580 (P<0.05).5. The results of EMSA showed that, after AML BMSCs incubated with tryptase for 48h, compared with the control group, the activation of NF-κB were increased, and the increasing was significantly reduced by PDTC (P<0.05).6. The results of Real time RT-PCR and Western-blot showed that after AML BMSCs incubated with PAR-2,ERK,p38MAPK and NF-κB specificity blocking agent respectively (FLLSY-NH2, PD98059, SB230580 and PDTC) treated cells for 30 min, VEGF mRNA and protein expression which increased by tryptase were reduced (P <0.05).Conclusions1. The reorganized tryptase could promote VEGF overexpression in AML BMSCs, showing a dose-dependent relationship in a certain range.2. Tryptase secreted by leukemia cell line U937 can also promote VEGF highly expressed in AML BMSCs.3. PAR-2 were expressed on BMSCs surface, playing an important role in tryptase increaseing the expression of VEGF in AML BMSCs4. ERK, p38MAPK and NF-κB signaling pathway were involved in the tryptase increasing VEGF expression in AML BMSCs. |
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