ABSTRACT
OBJECTIVE: To investigate the effect of autophagy gene Beclin1 silencing on enhancing the drug sensitivity of imatinib-resistant human myeloid leukemia cell line K562/IMA. METHODS: Imatinib-resistant human myeloid leukemia cell line K562/IMA was constructed by the combination of the first dose of high-dose shock therapy and gradual increase of dose. Imatinib interfered with K562 and K562/IMA cell lines. Cell viability was detected by CCK-8. Apoptosis rate was detected by flow cytometry. Western-blot assay was used to determine the drug resistance level. The expression of Beclin1 in drug resistant strains was detected. Small interfering RNA (siRNA) transfected Beclin1 siRNA-Beclin1 was used to silence the Beclin1 gene of K562/IMA. Control group (control), siRNA negative control group (siRNA-NC) and Beclin1 siRNA group (siRNA-Beclin1) were set up. RT-QPCR and Western-blot were used to identify the expression level of Beclin1 protein and mRNA in each group after silencing. CCK-8 method was used to detect the sensitivity of each group of cells to imatinib, and the half inhibitory concentration IC50 was calculated. The apoptosis rate of each group was detected by flow cytometry. The expression levels of Beclin 1, Bcl-2, Bax, cleaved-caspase 3, caspase-3 and cytochrome C(cyto-C) were detected by Western-blot assay, and the metastatic and invasive ability of cells was detected by Transwell chamber. RESULTS: K562 / IMA cell lines were resistant to imatinib at 10 μmol•L-1, and the expression of Beclin1 was higher than that of K562 cell lines. The cell viability of K562/IMA was significantly higher than that of K562 cell lines in the gradient concentration range, while the apoptosis rate was lower than that of K562 cell lines. After siRNA silencing Beclin-1, the cell viability and apoptosis rate of siRNA-Beclin 1 group were significantly lower than those of control group, and the IC50 value of half inhibitory concentration was significantly lower than that of control group. The levels of Bax, cleaved-caspase 3, caspase-3 and cyto-C in the cells were significantly higher than those in the control group, while the levels of Bcl-2 were significantly lower than those in the control group. CONCLUSION: Beclin1 plays an important role in imatinib resistance in human myeloid leukemia, and autophagy may be involved in the drug resistance process. After silencing Beclin1, cell resistance level can be reduced and drug sensitivity improved.
ABSTRACT
AIM:To investigate the effects of DL-3-n-butylphthalidle (NBP) on angiogenesis of human um-bilical vein endothelial cells ( HUVECs) and the role of vascular endothelial growth factor ( VEGF)/VEGF receptor 2 (VEGFR2)-Notch1/Delta-like ligand 4 (Dll4) signaling pathway in this process. METHODS:The serum-free medium and anoxic tank were used to simulate the conditions of hypoxia and ischemia ( H/I). HUVECs were divided into control group, H/I group, H/I+NBPhigh group and H/I+NBPlow group. The HUVECs in control group were conventionally cul-tured, and those in H/I group were cultured under H/I intervention. The HUVECs in H/I+NBPhigh group were treated with NBP at 20 μmol/L under H/I intervention. The HUVECs in H/I+NBPlow group were treated with NBP at 5 μmol/L under H/I intervention. The cell viability of each group was measured by CCK-8 assay. The migration ability of the HUVECs in each group was detected by cell scratch test. The vessel formation ability of the HUVECs was examined by in vitro angiogenesis assay. The expression of VEGFR2, Notch1 and Dll4 at mRNA and protein levels was determined by qPCR and Western blot, and the expression of VEGF was determined by qPCR and ELISA. RESULTS:NBP increased the viability of HUVECs, and promoted the migration ability and the formation of blood vessels in vitro under H/I interven-tion. These effects of NBP at high dose were more significant than those at low dose. NBP increased the expression of VEGF, VEGFR2, Notch1 and Dll4 at mRNA and protein levels (P<0.05). CONCLUSION:NBP promotes HUVECs to form blood vessels under H/I intervention. The mechanism may be related to the activation of VEGF/VEGFR2-Notch1/Dll4 signaling pathway.
ABSTRACT
AIM:To detect the myeloid-derived suppressor cells(MDSCs )in peripheral blood from the pa-tients with Parkinson disease(PD)and its clinical significance.METHODS:The patients(n=80)diagnosed PD from January 2016 to March 2017 in our hospital and 20 healthy volunteers were selected as the subjects.According to the Hoehn-Yahr staging,80 PD patients were staged,of whom 22 were Ⅰ,24 were Ⅱ,20 were Ⅲ,14 were Ⅳ,and 0 was Ⅴ. Peripheral blood(5 mL)samples from the patients with PD and the healthy volunteers were collected and the mononuclear cells were isolated.The levels of CD14 +CD11b+cells and CD14 -CD11b+cells in the peripheral blood were detected by flow cytometry.The two populations of the cells were sorted by magnetic beads.The mRNA levels of arginase 1(ARG1),interleukin-10(IL-10)and cyclooxygenase 2(COX-2)were detected by qPCR.The expression of surface membrane pro-teins CD14 and CD11b,and immunosuppressive factors ARG1,IL-10 and COX-2 was determined by Western blot and ELISA.RESULTS:No significant change of CD14 +CD11b+cells between the patients with PD and normal controls was observed,but the cells with CD14 -CD11b+increased significantly in the patients with PD compared with the control peo-ple(P<0.05).The CD14 -CD11b+cells in peripheral blood of the patients were related to the stage of Hoehn-Yahr.The CD14-CD11b+and CD14+CD11b+cells showed high levels of IL-10 and COX-2,and the high level of ARG1 was only expressed in the CD14 -CD11b+cells.The expression of ARG1 in the CD14 -CD11b+population from PD patients was significantly different from that of CD14+CD11b+population and normal subjects(P<0.05).CONCLUSION:The CD14-CD11b+cells and ARG1 expression level in peripheral blood of the PD patients can be used to evaluate the patho -genesis and staging.Immunosuppression may play an important role in the pathogenesis and development of PD.