Expression of MicroRNAs in Peripheral Blood of Patients with Primary Immune Thrombocytopenia and Its Correlation with the Imbalance of Th1/Th2 Cell / 中国实验血液学杂志

OBJECTIVE@#To investigate the expressions of microRNAs in peripheral blood of patients with primary immune thrombocytopenia(ITP) and its correlation with the imbalance of Th1/Th2 cells.@*METHODS@#Thirty patients with ITP (ITP group) and 15 healthy people (control group) were enrolled．Real-time polymerase chain reaction (RT-PCR) was used to detect the expressions of six miRNAs (miR-107,miR-205-5p,miR-138-5p,miR-326,miR-1827,miR-185-5p) and Th1-specific transcription factor T-bet mRNA and Th2-specific transcription factor GATA-3 mRNA in the peripheral blood of the two groups. Th1 and Th2 cells were detected by flow cytometry. The expressions of Th1-cytokines TNF-α and IFN-γ and Th2-cytokines IL-4 and IL-10 were detected by AimPlex multiple immunoassays for Flow. The expression difference of miRNAs, mRNA, Th1, Th2 cells and cytokines of the two groups were compared, and the correlations of miRNAs to mRNA, Th1, Th2 cells and cytokines were analyzed in ITP group.@*RESULTS@#The expressions of miRNAs（miR-107, miR-205-5p, miR-138-5p, miR-326, miR-1827, miR-185-5p）and Th2-specific transcription factor GATA-3 mRNA of the patients in ITP group were significantly decreased (P<0.05) as compared with those in control, while the expressions of Th1 cells and Th1-specific transcription factor T-bet mRNA and Th1-cytokines TNF-α were significantly increased (P<0.05), also for the ratios of T-bet mRNA/GATA-3 mRNA and Th1/Th2 cells were significantly increased (P<0.05). The relative expressions of miR-107, miR-205-5p, miR-138-5p in ITP patients were negatively correlated with Th2 cells (r=-0.411, r=-0.593, r=-0.403,P<0.05) and the relative expression of miR-1827 was negatively correlated with TNF-α (r=-0.390).@*CONCLUSION@#The relative expressions of the six miRNAs in peripheral blood of patients with ITP are significantly decreased, which result in the increasing ratio of T-bet mRNA/GATA-3 mRNA, then lead to the imbalance of Th1/Th2.

Detection of Exosomal PML-RARA Fusion Gene Expression Level by Droplet Digital PCR / 中国实验血液学杂志

OBJECTIVE@#To establish a method for detecting the exosomal PML-RARA fusion gene expression by droplet digital PCR (ddPCR).@*METHODS@#By using Taqman probe-based ddPCR technique, the method that able to detect both long and short isoforms of PML-RARA fusion gene transcripts was established. RNA from PML-RARA negative cell line HL-60 as negative control was used to set the limit of blank (LOB), while the RNA from PML-RARA positive cell line NB4 and the recombinant plasmid pSG5-PML-RARA(S) were used to set the limit of detection (LOD) for long and short PML-RARA transcripts, respectively. Furtherly, the expression of exosomal PML-RARA fusion gene in NB4 cell culture supernatant and serum of patients with acute promyelocytic leukemia (APL) was analyzed by ddPCR technique.@*RESULTS@#The LOB of ddPCR assay for long and short PML-RARA transcripts were 0.0725 and 0.083 copies per microliter of PCR reaction system, respectively, while the LOD of long and short PML-RARA transcripts were 0.19 and 0.21 copies per microliter of PCR reaction system, respectively. In addition, the expression of exosomal PML-RARA fusion gene derived from both NB4 cell culture supernatant and serum of APL patients was successfully detected.@*CONCLUSION@#A ddPCR-based technique for detecting fusion gene transcripts has been established, which can be used to analyze absolute quantification in the minimal quantity of PML-RARA transcripts derived from exosomes. It suggests the possibility of this technique to non-invasively and dynamicly monitore the exosomal PML-RARA transcripts from APL patients' serum.

Level of Regulatory B Cells in Patients with Immune Thrombocytopenia and Its Clinical Significance / 中国实验血液学杂志

OBJECTIVE@#To investigate the role of regulatory B cells (Breg) in pathogenesis of immune thrombocytopenia(ITP) and its clinical significance.@*METHODS@#A total of 40 ITP patients and 20 normal controls were enrolled in this study. The content of Breg, Th1, Th2, Th17 and Treg cells were detected by flow cytometry (FCM). The expression level of IL-10,TGF-β, CD40 and CD40L was detected by AimPlex Flow High Throughput Screening Technology.@*RESULTS@#The of Breg cells in ITP patients was significantly lower than that in normal controls (P<0.05)，the expression levels of IL-10,TGF-β and CD40L in ITP patients were also significantly lower than those in normal controls (P<0.05). The contents of Th1 cells in ITP patients were significantly higher than that in normal controls (P<0.05), whereas the contents of Th2, Th17 and Treg cells in ITP patients were significantly lower than those in normal controls (P<0.05).@*CONCLUSION@#The Breg cells may play an important role in the pathogenesis of ITP.

Secretory Imbalance between Pro-inflammatory and Anti-inflammatory Cytokines in the Patients with Immune Thrombocytopenia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the imbalance of pro-inflammatory and anti-inflammatory cytokines in the patients of immune thrombocytopenia (ITP).</p><p><b>METHODS</b>Thirty-five patients with ITP were enrolled in ITP group, while 28 healthy persons were included in control group. The expressions of IL-8, IL-17A, IL-22, TNF-α, IFN-γ, IL-4, CD40, CD40L, TGF-β and IL-10 were detected by flow cytometry with aimPlex multiple immunoassay Flow.</p><p><b>RESULTS</b>The expressions of pro-inflammatory factors IL-8, IL-17A, IL-22, IFN-γ and TNF-α in ITP group all were significantly higher than that in the control group (P<0.05), however the expressions of anti-inflammatory factors IL-4, CD40L, TGF-β and IL-10 in ITP group all were significantly lower than that in the control group (P<0.05). The expression of CD40 was not significantly different between ITP group and control group (P>0.05). Expressions of TNF-α significantly related with platelet counts in both ITP group and control group (ITP group, r=0.64, P<0.05; control group, r=-0.41, P<0.05). However the expression of CD40, TGF-β, CD40L, IL-8, IL-17A, IL-22, IL-10, IL-1β, IFN-γ and IL-4 significantly did not relate with platelet counts in both ITP and control group.</p><p><b>CONCLUSIONS</b>The secretory imbalance between pro-inflammatory and anti-inflammatory cytokines exists in the patients of ITP. The decrease of Plt regulated may be regulated by the abnormal expression of TNF-α.</p>