Relationship between the Expression of miRNA181a-5p and the Imbalance of Treg/Th17 in Primary Immune Thrombocytopenia / 中国实验血液学杂志

OBJECTIVE@#To investigate the role of relationship between the expression of miRNA181a-5p and imbalance of Treg/Th17 in the pathogenesis of primary immune thrombocytopenia(ITP), which contributes to clarify the mechanism of T cell immune imbalance in ITP patients.@*METHODS@#Peripheral blood was collected from 37 ITP patients, concluding 21 untreated patients and 16 effectively treated patients, and 19 healthy controls; Peripheral blood mononuclear cells (PBMC) were isolated and the expression of miRNA181a-5p and Notch1 was analyzed by RT-PCR. The proportion of Th17 subsets and Treg cells in the peripheral circulation was detected by flow cytometer (FCM). Clinical data of ITP group was collected, including age, platelet count and disease course.@*RESULTS@#The expression of miR-181a-5p was significantly decreased in ITP group than that of healthy control group (P＜0.01). After effective treatment, the expression of miR-181a-5p was significantly higher than that of ITP group (P＜0.05), but still significantly lower than that of healthy control group (P＜0.01); The expression of Notch1 was significantly increased in ITP group and effectively treated group than that of healthy control group (P＜0.01). There was no significant difference in proportion of Treg cells in ITP group, effectively treated group and healthy control group (P＞0.05). The proportion of Th17 subsets in ITP group was significantly increased than that of healthy control group (P＜0.05), while the ratio of Treg/Th17 was significantly decreased (P＜0.05). There was a positive correlation between the expression of miR-181a-5p and ratio of Treg/Th17 in ITP group (r=0.555).@*CONCLUSION@#The expression of miR-181a-5p is significantly decreased in ITP patients, which is closely related to the imbalance of Treg/Th17 cells. After effective treatment, the expression of miR-181a-5p can be significantly corrected, but still failed to reach the level of healthy people. While the expression of Notch1 is significantly increased in ITP patients, and could not reach the level of healthy people after effective treatment.

Analysis of miRNA differential expression in peripheral blood cells of primary immune thrombocytopenia patients / 中国实验血液学杂志

This study was purposed to clarify the difference of microRNA (miRNA) expression in the peripheral blood cells of patients with primary immune thrombocytopenia (ITP) and normal controls. Exqion miRCURY(TM) microarray was used to investigate differentially expressed miRNA of peripheral blood cells obtained from affected ITP patients and the healthy controls. Cluster analysis was used to identify miRNA expression profile between the ITP patients and the healthy controls. Real-time PCR was used for validation. The results showed that a total of 159 miRNA were found to be differentially expressed in ITP patients compared to the controls, with 79 up-regulated and 80 down-regulated. Based on these differentially expressed miRNA, a tree with clear distinction between the controls and ITP patients was generated by cluster analysis. Real-time PCR confirmed microarray analysis results. It is concluded that differentially expressed miRNA were found in the peripheral blood cells from ITP patients, which may be potential novel biomarkers for ITP as well as help to elucidate pathogenic mechanisms of ITP.

Analysis of clinical features and genotype in three Chinese pedigrees with Glanzmann thrombasthenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the clinical feature and alpha II b beta 3 gene mutations of three Glanzmann thrombasthenia (GT) pedigrees.</p><p><b>METHODS</b>Platelet counts (BPC), blood film, bleeding time, platelet aggregation and flow cytometry were used for phenotype diagnosis of all the patients. All the exons of alpha II b and beta 3 genes were amplified by polymerase chain reaction (PCR) and direct sequencing was performed for mutational screening. One hundred and three healthy blood donors were as normal controls.</p><p><b>RESULTS</b>Three probands showed normal BPC, defective platelets aggregation, prolonged bleeding time and significantly reduced platelet aggregation to ADP, epinephrine, and collagen, while relatively normal aggregation to ristocetin. Flow cytometry showed platelet surface expressed alpha II b beta 3 was strongly reduced in proband 1 and proband 3 and mildly reduced in the amount of surface expressed alpha II b beta 3 (63%) in proband 2. Sequencing results showed that proband 1 had a G10A homozygous mutation in alpha II b, and a G1412T homozygous mutation in beta3. Compound heterozygous mutations in beta3, G1199A and 1525delC were identified in proband 2. No mutations in alpha II b beta 3 gene were identified in proband 3.</p><p><b>CONCLUSIONS</b>Compound homozygous mutations, GI0A in alpha II b and G1412T in beta3, lead to GT in proband 1. Compound heterozygous mutations in beta3, G1199A and 1525delC, lead to GT in proband 2. The mutations of G10A, G1412T and 1525delC were reported for the first time in GT patients.</p>

Molecular mechanisms of Glanzmann thrombasthenia caused by alpha II b L721R and Q860X compound heterozygous mutation / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the molecular mechanisms of Glanzmann thrombasthenia caused by alpha II b L721R and Q860X compound heterozygous mutation.</p><p><b>METHODS</b>All exons and exon-intron boundaries of alpha II b and beta3 gene were amplified by PCR and analyzed by direct DNA sequencing. Gene polymorphisms were excluded by direct DNA sequencing. Alpha II b L721R and Q860X mutants expressing vectors were constructed by in vitro site-directed mutagenesis. The expression of alpha II b L721R and Q860X mutants on transfected cell membrane were analyzed by flow cytometry and the whole expression level was confirmed by Western blot. The subcellular localizations of alpha II b L721R and Q860X mutants were determined by immunofluorescent confocal scanning microscopy.</p><p><b>RESULTS</b>The alpha II b compound heterozygous mutations, T2255G (L721R) and C2671T (Q860X), were identified in the proband, the former being inherited from the maternal side and the latter the paternal side. The 293T cells cotransfected with mutated alpha II b L721R and wild-type beta3 expression plasmids expressed 2.1% of normal amount of alpha II b on the cell surface as shown by FACS, in contrast to 31.9% of normal amount of alpha II b on the cells cotransfected with cDNAs of mutated alpha II b Q860X and wildtype beta3 expression plasmids. Western blot of the cell lysates showed no detectable mature alpha II b in cells lysates with L721R mutant. While, truncated alpha II b protein was detected in cell lystes with Q860X mutant. Immunofluorescence studies demonstrated that both L721R and Q860X mutant pro-alpha II bbeta33 complex colocalized in endoplasmic reticulum, but a little in Golgi.</p><p><b>CONCLUSIONS</b>The L721R and Q860X mutations of alpha II b prevent transport of the pro-alpha II bbeta3 complex from the endoplasmic reticulum to the Golgi, hindering its maturation and surface expression. The impaired alpha II bbeta3 transport is responsible for the thrombasthenia.</p>

Antithrombotic mechanisms of holothurian glycosaminoglycan extracted from sea cucumber / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the antithrombotic mechanisms of holothurian glycosaminoglycan (GAG) extracted from sea cucumber.</p><p><b>METHODS</b>Human endothelial cell line EA. hy926 cells were treated with 10 mg/L GAG or 10U/mL unfractionated heparin (UFH) by short-term (15 min - 2 h) and longer-time incubation (6 h - 48 h). Different doses of GAG were used to stimulate EA. hy926. Released free tissue factor pathway inhibitor(TFPI) was determined by ELISA assay. TFPI expression was investigated by immunofluorescent method and TFPI mRNA level by real-time PCR. In a 96-wells microtitre plate, pooled normal plasma containing different concentrations of GAG was allowed to clot by addition of thrombin and calcium chloride, fibrinolysis was induced by addition of t-PA. TRR (TAFI-related retardation of clot lysis) was used to assess thrombin-activatable fibrinolysis inhibitor(TAFI) functional activity.</p><p><b>RESULTS</b>GAG increased TFPI synthesis, expression and secretion in a dose- and time dependent manner. GAG at low concentrations could lengthen while at intermediate concentrations could shorten clot lysis times significantly as compared to control values. TRR was dose-dependently decreased on addition of GAG.</p><p><b>CONCLUSIONS</b>GAG increases TFPI synthesis, expression and secretion of endothelial cells. GAG at intermediate concentrations significantly affects clot stability of a developing clot by means of diminishing TAFI activation.</p>