Effects of ICAT silencing in Wnt signaling pathway and NSC67657 induced cell differentiation of HL-60 cells / 中华血液学杂志

Objectives@#To investigate the effect of β-catenin interacting protein 1 (ICAT) silence in Wnt signaling pathway and sterol drug NSC67657 induced cell differentiation of HL60 cell.@*Methods@#HL-60 cells were treated with NSC67657, the cell surface antigen CD14 expression was detected by flow cytometry. Lentivirus LV-ICAT-RNAi vector was constructed and infected HL-60 cells. Then the ICAT gene and protein expression were analyzed using real-time qPCR and Western blot technique. Furthermore, Co-immunoprecipitation assay was used to confirm the interaction of β-catenin/ICAT proteins, and Western blot was employed to compare the expressions of Wnt signaling pathway downstream targets Cyclin D1, TCF-1 and c-Jun between Lentivirus LV-ICAT-RNAi vector infected HL-60 (HL-60i) cells and un-infected HL-60 (HL-60v) cells. The cellular differentiation of HL-60i and HL-60v cells treated with NSC67657 for 24 h was evaluated by Wright’s staining, transmission electron microscopy and flow cytometry analysis.@*Results@#HL-60 cells could be induced to differentiate into monocytes by 10 μmol/L NSC67657. The CD14 positive cells could reach to (92.30±5.14) % after NSC67657 treatment for 5d. The co- immunoprecipitation assay demonstrated that ICAT protein did interact with β-catenin protein, and the absorbance of protein electrophoresis bands increased in differentiated cells. The expressions of Wnt signaling pathway downstream target proteins in HL-60i cells were higher than that in HL-60v cells when they were treated by 10 μmol/L NSC67657, but lower than NSC67657 untreated cells. CD14 positive HL-60i cells were significantly lower than that of HL-60v cells[ (8.33±3.14) % vs (19.08±4.73) %]when treated with NSC67657, but still higher than that of uninfected and untreated HL60 cells[ (0.60±0.03) %] (F=119.24, P=0.010) . The results of cellular morphology and ultrastructure observation were also in accord with that of cell surface antigen analysis.@*Conclusions@#ICAT does participate in HL-60 cells monocytic differentiation induced by NSC67657, and Wnt/β-catenin signaling pathway might play a bridge role.

Establishment and preliminary application of detection of Mycobacterium tuberculosis in sputum based on variable number tandem repeat / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a laboratory method for detection of Mycobacterium tuberculosis in sputum based on variable number tandem repeat (VNTR).</p><p><b>METHODS</b>Mycobacterium tuberculosis was tested by VNTR and fluorescent quantitative reverse transcription polymerase chain reaction (FQ-PCR) in 130 sputum samples from patients with pulmonary tuberculosis and 200 specimens from patients with other lung diseases. According to the amplification conditions and clinical detection needs, MTUB21, MUTB04, QUB18, QUB26, QUB11b, MIRU31, MIRU10 and MIRU26 were selected as test targets. The results of VNTR and FQ-PCR were compared with Lowenstein-Jensen culture and clinical diagnosis, and analyzed by chi-square test.</p><p><b>RESULTS</b>With the results of L-J culture as the standard, the sensitivity and specificity of VNTR were 93.1% (108/116) and 97.7% (209/214), and those of FQ-PCR were 94.0% (109/116) and 96.7% (207/214), respectively; no significant difference was observed between two groups (χ2=0.352, P=0.569). Using the clinical diagnosis as the standard, the sensitivity and specificity of VNTR were 86.9% (113/130) and 100% (200/200), and those of FQ-PCR were 87.7% (114/130) and 99.0% (198/200), respectively; the difference was not statistically significant (χ2=0.030, P=0.862). In 113 VNTR positive samples, the molecular codes differed from each other in 98.2% samples (111/113); only 2 samples had identical code (5-4-6-8-5-5-3-8).</p><p><b>CONCLUSION</b>The study suggests that VNTR provides a promising method for diagnosis of clinical tuberculosis.</p>

Influence of leptin on secretion of chemokine in THP1 cells and its mechanism / 吉林大学学报(医学版)

Objective To investigate the effect of leptin on the secretion of chemokine in THP1 cells and explore its related mechanism, and to provide basis for research on the role of leptin in immune response.Methods The expressions of Ob-Rb and Ob-Rt in THP1 cells were detected by RT-PCR and flow cytometry (FCM).The THP1 cells at logarithm growth phase were selected and randomly divided into blank control group and different concentrations(10,50,100,200μg· L-1 )of leptin groups.Transwell chamber assay was performed to detect the number of invated THP1 cells.The THP1 cells were divided into blank control group and 100μg·L-1 leptin group.Western blotting method was carried out to detect the expressions of p-AKT,p-ERK 1/2,and p-STAT3 in THP1 cells.The THP1 cells were divided into blank control group and 100μg·L-1 leptin group,100μg·L-1 leptin+ DMSO group,100μg·L-1 leptin+50μmol·L-1 AG490 group,100μg·L-1 leptin+10μmol·L-1 LY294002 group and 100μg·L-1 leptin+ 10 mol·L-1 PD980590 group.RT-PCR and Western blotting methods were performed to detect the expression of IL-8.Results Ob-Rb and Ob-Rt were highly expressed in THP1 cells. Compared with blank control group,the number of invated THP1 cells was significantly increased in 50,100,and 200μg·L-1 leptin groups (P0.05).Conclusion leptin can up-regulate the expression of chemokine in THP1 cells,which might be associated with PI3K-AKT and MAPK/ERK 1/2 signaling pathways.