Regulation Of ITP Plasma In Megakaryocytopoiesis

Background and ObjectivesIdiopathic thrombocytopenic purpura(ITP) is an common hemorrhagic diseases characterized by a low platelet count and normal or increased number of immature megakaryocytes in bone marrow.The pathogenetic mechanism of ITP is not completely clear yet. Both autoantibodies to platelet glycoproteins (GP) and viral infections attribute to the thrombocytopenia.For a long time, Autoantibodies to platelet glycoproteins (GP) Ⅱb/Ⅲa and/or Ib/IX have been considered as the main causes of ITP. Platelets sensitized by autoantibodies or antibody-containing immune complexes are cleared by phagocytic cells in the reticuloendothelial system, leading to clinical symptoms such as petechiae or bleedings. But,platelet destruction is not the only mechanism of thrombopenia,as two-thirds of the patients,of which the platelet turnover has been be discovered reduced or nomal.So,ITP has been involved not only platelet destruction, but also deficits in platelet production.As the precursors of platelets. megakaryocytes also express GPIIb/IIIa and/or GPIb/IX,which can be binded by the autoantibodies resuting in the destruction of megakaryocytes.Studies showed that autoantibodies against either platelet GPIb or platelet GPIIb/IIIa in ITP plasma are involved in megakaryocytic destruction in vitro. But the amounts of megakaryocytes were usually nomal or increased in clinical. So,the thrombocytopenia shoule not be attributed to the decreasing of megakaryocytes.Moreover, Morphology study showed that,these megakaryocytes were generally immature.Megakaryocytopoiesis is governed by a complex network of haematopoietic growth factors that regulating the different stages of the process,.Any exception occurs in which will affect the development of megakaryocytes. MicroRNAs are a class of endogenous non-coding RNA molecules with about22length which can block gene expression through base-pairing to partially, or completely, sequence-homologous regions within the3’untranslated regions (UTR) of mRNAs. They may participate in megakaryocytopoiesis by regulating the transcription factors involved in the development of megakaryocytes.The same miRNA may target different genes,or the same gene may be targeted by different miRNAs.Part of patients with acute ITP have a history of an infectious illness a few days to a few weeks before the onset of thrombocytopenia. Widely different viruses, including herpes simplex virus,Epstein-Barr virus, influenza, varicella zoster virus, cytomegalovirus, mumps,parvovirus, and rubella virus have been suspected as potential triggers of the disease. At present,miRNAs have been identified in all multi-cellular eukaryotes, some unicellular eukaryotes and even acellular organism,e.g.virus. Viral miRNAs were discovered only recently, and functional relationships between viruses,viral or cellular miRNAs and viral or cellular mRNA are only now beginning to be elucidated.Studie showed that viral miRNAs may change the expression of mRNAs of both host cells and their own.While,the changed gene expression of cellular miRNAs also directly or indirectly promote of inhibit virus replication and pathogenesis conversely. Some reports said that viral miRNAs could play a key role in tumorigenesis.However,whether there is a connection between viral miRNAs and ITP by regulating megakaryocytopoiesis is unknown yet.So,it is very important to explore the expression of viral miRNAs in megakaryocytes. viral miRNAs may provide new therapy for ITP.MethodsPartⅠ In vitro expansion of megakaryocytes from umbilical cord blood(CB) mononuclear cells(MNCs)All CB samples,collected in sodium heparin (20IU/mL)tubes were taken from consenting women who had normal, full-term pregnancies with no complications. Umbilical cord blood mononuclear cells were separated by density gradient centrifugation. Prepare the cytokines,including recombinant human stem cell factor(rhSCF,SCF), recombinant human thrombopoietin(rhTPO,TPO), recombinant human interleukin(rhIL-3,IL-3) and recombinant human interleukin(rhIL-6,IL-6). The MNCs were cultured at5*105/ml in24-well cell culture plates for up to10days in IMDM medium supplemented with10%fetal calf serum,2mM L-Glutamine and recombination growth factors, i.e., SCF(50ng/ml)+TPO (20ng/ml)+IL-3(10ng/ml)+IL-6(lOng/ml). Three groups were divided according to the combination of cytokines,including A:SCF+TPO+IL-3,B:SCF+TPO+L-6and C:SCF+TPO+IL-3+IL-6.At day0and at3-day intervals thereafter, the cultures were boosted with the same combination of recombination growth factors. Cultures were incubated at37℃in a humidified atmosphere of5%CO2for10days. Morphous of megakaryocytes was observed with an inverted microscope and light microscope. The quantity of megakaryocytic cells (CD41a+cells) in culture was counted and flow cytometric analysis was performed then.Part Ⅱ Effects of ITP plasma on megakaryocytopoiesis ITP plasma samples from24randomly selected ITP patients were collected for analysis.10healthy volunteers were examined as controls. Plasma was obtained from EDTA-anticoagulated blood by centrifugation. Plasma was added to medium at the beginning of megakaryocytes expansion.The cells were collected after10days later. Morphous of megakaryocytes was observed under an inverted microscope and light microscope; ultrastructure was observed under a electron microscope; quantity of CD41a+cells was detected by flow cytometry;miRNAs expression was analyzed by microarray assay;QRT-PCR was performed to confirm the results of microarray analysis.PartⅢ Viral miRNAs and ITPSelected6viral miRNAs,including ebv-miR-BART6-3p ebv-miR-BART19-3p、hsvl-miR-H7*、hsvl-miR-H8*、hsvl-miR-H14-3p and hsv2-miR-H7-3p according to the results of microarray analysis that were upregulated in megakaryocytes incubated with ITP plasma. QRT-PCR was performed to confirm the results of microarray analysis.Statistical methods:The analysis was performed using SPSS13.0software package. The data was represented as the mean±standard deviation (X±S). Differences among prodction of megakaryocytes in three groups of cytokine combinations measured by flow cytometry were determined using One-way ANOVE. Mann-Whitney U noparametric tests was performed between the two groups of megakaryocytes incubated with ITP or nomal plama.One-sample t-test was performed to compare the fold change of miR-146a in the ITP group of megakaryocytes and plasma. The fold change of the six viral miRNAs in the ITP group of megakaryocytes was analyzed using One-sample t-test.P values less than0.05are considered statistically significant.Results PartⅠ In vitro expansion of megakaryocytes from umbilical cord blood(CB) mononuclear cells(MNCs)1. Morphology of megakaryocytes under inverted microscope Three groups of megakaryocytes were morphologically similar to normal megakaryocytes at day10of culture.But the number of megakaryocytes in C group is up-regulation obviously than A and B.2. Megakaryocytic morphous under light microscope The cells are large with irregular shape of nucleus and incarnadine cytoplasm,the cell membrane of which is unclear accompany with extending pseudopodium.3. Quantity of the three group megakaryocytes was detected by flow cytometry Megakaryocytes were defined by fluorescence-activated cell sorter (FACS) analysis as CD41a+cells. One-way ANOVE was performed to detect the difference of percentage of CD41a+cells.The percentage of megakaryocytes in C was36.03%±0.27%,which upregulated significantly compared with A (10.23±0.21)%and B(10.10±0.15)%.There was statistical difference (F=14673.22, P<0.001) among the three groups.Multiple comparison amongA、B and C was analyzed using Bonferroni Multiple Comparisons.There were significantly statistical differences between C and A(P<0.001) or B(P<0.001),but there is no statistical difference between A and B(P=1.000).Part II Effects of ITP plasma on megakaryocytopoiesis1. Two group megakaryocytes under inverted microscope and light microscope Megakaryocytes cultured in media containing ITP plasma were morphologically similar to normal megakaryocytes at day10of culture.But the number of megakaryocytes incubated with ITP plasma were up-regulation obviously than the normal megakaryocytes under inverted microscope. And the thrombopoiesis was derangement in the ITP group under light microscope.2. Ultrastructure was observed under a electron microscope The ultramicrostructure of megakaryocytes were obviously different between two groups. For ITP group, the nuclear cytoplasm ratio of megakaryocyte is higher, although the diameter of cell is smaller, the cytoplasmic ribosome was more abundant, but the cytoplasm platelet demarcation membrane system was immature. The normal megakaryocyte looks like honeycomb.3. Quantity of the two group megakaryocytes was detected by flow cytometry Megakaryocytes were defined by fluorescence-activated cell sorter (FACS) analysis as CD41a+cells. Mann-Whitney U noparametric tests was performed between the two groups of megakaryocytes incubated with ITP or nomal plama.Percentage of megakaryocytes cultured with24ITP plasma was (11.89±1.62)%,which significantly lower than the contral(33.61±3.24)%. There were significantly statistical differences between them(Z=-4.536, P<0.001).4. MiRNAs are differentially expressed between the two groups of megakaryocytes analyzed by microarray To identify specific miRNAs that might function in megakaryocytopoiesis, we analyzed global miRNA expression using miRCURY LNA Array that covered all microRNAs in miRBase.89miRNAs were significantly upregulated in the ITP group,such as hsa-miR-101, hsa-miR-124*, hsa-miR-30c-1*, hsa-miR-146a and so on. Interestingly,14viral miRNAs, including hsv2-miR-H9-3p, hsvl-miR-H7*, ebv-miR-BART19-3p, hsv2-miR-H24, hsv2-miR-H10, hsvl-miR-H17, hsvl-miR-H8*, ebv-miR-BART6-3p, hsvl-miR-H14-3p, ebv-miR-BART8*, sv40-miR-S1-5p, ebv-miR-BHRF1-2, hsv2-miR-H7-3p and hsv2-miR-H6were elevated in megakaryocytes incubated with ITP plasma.63miRNAs such as hsa-miR-17, hsa-miR-106a, hsa-miR-106b*, hsa-miR-126*and hsa-miR-128were dereased in the ITP group.But there is no viral miRNAs decreased in the ITP group.5. QRT-PCR was performed to confirm the results of microarray analysis MiR-146a was selected to verified by QRT-PCR.The expression of miR-146a was calculated by2-ΔΔCt,while the expression of miR-146a in megakaryocytes incubated with ITP plasma was increased3.71±0.65-fold which was statisticly difference with the contral(t=20.549,P<0.001).The expression levels of miR-146a in the ITP plasma were increased3.06±0.75-fold which were statisticly difference with the contral(t=13.430,P<0.001).PartⅢ Viral miRNAs and ITPQRT-PCR was used to validate the selected6miRNAs significantly altered from the miRNA microarray results.While all the6viral miRNAs showed an increased expression of more than one-fold, which were consistent with the miRNA microarray results The relative expression of each viral miRNA between megakaryocytes incubated with ITP and nomal plasma described using the2-ΔΔCt.While the5of the6viral miRNAs was statistically different between the two groups of megakaryocytes except ebv-miR-BART6-3p (P=0.073).Conlusion1. The combination of SCF+TPO+IL-3+IL-6was efficient for the expansion of megakaryocytes2. ITP plasmas not only reduced the total number of megakaryocytes produced during the culture period but also inhibited megakaryocyte maturation.3. The expression of miRNAs was different obviously between the two groups of megakaryocytes incubated with ITP and nomal plama.4. Viral miRNAs may participate in pathogenesis of ITP by regulating megakaryocytopoiesis.The innovation of this study 1. To explore the different expression of miRNAs between the mature and inmature megakaryocytes,we used microarray assay by adding ITP or nomal plasma to the cultures of megakaryocytes respectively.2. Our study primarily discussed the relationship between viral miRNAs megakaryocytopoiesis and ITP.