| PART 1. The Study of the Development of Megakaryocytes Modulated by Viral microRNAObjective To study the effect of viral ebv-miR-BART6-3p on the proliferation, differentiation and development of megakaryocytes, and to investigate the probable mechanism.Method(?) The mononuclear cells were collected from 20 umbilical cord blood samples. CD34+ hematopoietic progenitor cells were isolated from the mononuclear cells and amplified into megakaryocytes.(?) Express vector of Ebv-miR-BART6 was constructed and transfected into CD34+ hematopoietic progenitor cell that was then cultured into megakaryocytes.(?) The difference of CD41+rates was compared between the groups by flow cytometric analysis and immunohistochemistry analysis. Megakaryocyte was identified.(?) The difference between groups of colony, morphology and the ultra microstructure of the megakaryocytes by inverted microscope, light microscope and electron microscope respectively.(?) The megakaryocytes were cultured with methylcellulose and MegaCult-C to test the ability of differentiation of every group.(?) The different of miR-185 expressed between the groups was analyzed through QRT-PCR.(?) The downstream target gene of miR-185 was predicted to be ABCG4 by Targetscan, and luciferase reporter validated it.(?) The effect of ebv-miR-BART6 on mRNA and protein expression of ABCG4 gene was detected by QRT-PCR and Western blot.Result(?) The viral vector of Ebv-miR-BART6-3p constructed and the blank vector (negative control) were transfected into CD34+hematopoietic progenitor cells isolated from the mononuclear cells derived from umbilical cord blood. The hematopoietic progenitor cells were cultured orientationally into megakaryocytes, and untreated group as blank control. The rate of transfection was more than 75% detected by flow cytometric analysis. CD41 expression rate of the megakaryocytes detected by flow cytometric analysis in ebv-miR-BART6-3p transfected group, blank control group and negative control group was 21.4±5.71%,36.2±4.09% and 32.7±6.83% respectively. CD41 expression rate of the megakaryocytes tested by immunohistochemistry in ebv-miR-BART6-3p transfected group, blank control group and negative control group wasl7.3±8.33%,33.7±6.27% and 30.3±5.83% respectively. Through light microscope, megakaryocytes were large, irregular nuclei, mature in blank control group and negative control group, while were micromegakaryocytes in ebv-miR-BART6-3p transfected group. More diploic structures and richer endoplasmic reticulum was observed in the two control groups than in the ebv-miR-BART6-3p group through transmission electron microscope. In the megakaryocytic colony experiment, there were 4.6±2.2 cells in each colony in the ebv-miR-BART6-3p group,16.3±3.7 cells in the blank control group and 14.7±4.3 cells in the negative control group. After 1000 cells were inoculated in 6-well plates, number of clones was 483 ± 51 in blank control group,407± 47 in negative control group and 217±33 in ebv-miR-BART6-3p group.(?) Normalizing the blank control group as 1, expression of miR-185-5p detected by QRT-PCR was down-regulated in ebv-miR-BART6-3p group and negative control group (0.323±0.034 vs 0.953±0.097).(?) There were two sites binding to 3’UTR of ABCG4 gene in the seed sequences of miR-185-5p. Luciferase reporter demonstrated that the luciferase activity of two-site wild-type ABCG4 decreased to 0.317±0.127, one-site mutated to 0.63±0.105, two-site mutated to 0.91±0.065.(?) Normalizing the blank control group as 1, expression of ABCG4 detected by QRT-PCR was up-regulated in ebv-miR-BART6-3p group and negative control group (3.327±0.687 vs 1.033±0.126). Expression of ABCG4 protein detected by Western blot was significantly higher than that of the control groups.Conclusion(?) Ebv-miR-BART6-3p inhibits megakaryocytes proliferation and differentiation.(?) Ebv-miR-BART6-3p can down-regulate miR-185-5p expression of megakaryocyte, and then enhance expression of its target gene, ABCG4.PART2.Hsa-miR-146 involves in the proliferation of megakaryocyte and correlates with prognosis of ITPObjective To confirm that hsa-miR-146 involves in the proliferation of megakaryocyte and correlates with prognosis of ITP.Method(?) The mononuclear cells were collected from 10 umbilical cord blood samples. CD34+ hematopoietic progenitor cells were isolated from the mononuclear cells and amplified into megakaryocytes, which were treated with 10uM dasatinib, according to the reference.(?) The difference of CD41+ rates was compared between the groups by flow cytometric analysis and immunohistochemistry analysis. Megakaryocyte was identified.(?) The difference between groups of morphology and the ultra microstructure of the megakaryocytes by light microscope and electron microscope respectively.(?) miRs expression of megakaryocytes was analyzed by miR microarray before and after treatment of dasatinib. Contrasting miRs expression profiles of megakaryocytes incubated with ITP plasma and with normal AB-type plasma, we found the difference of 8 kinds of has-miRs was more than 2.5 fold by cluster analysis.(?) To verify the microarray result, the plasma of 10 patients with ITP were collected and detected by QRT-PCR. The result of miR-146 was the most stable among the 8 miRs above mentioned, so we increased 46 more cases to analyze its dynamic change and the correlation with the prognosis.Results(?) Dasatinib enhances megakaryocyte proliferation and differentiation and inhibits platelet production. CD41 expression rate of the megakaryocytes detected by flow cytometric analysis in dasatinib group and control group was 40.8±3.22%, 36.2±4.09% and 32.6±4.71% respectively. CD41 expression rate of the megakaryocytes tested by immunohistochemistry in Dasatinib group and control group was 38.92±6.23% and 31.28±5.67 respectively. Through light microscope, megakaryocytes in control group were large, irregular nuclei, mature, surrounded by mature platelets clustering. Megakaryocytes in dasatinib group were similar to control group in size, nucleus and cytoplasm, but only a few immature giant platelet granules existed in the cytoplasm. More diploic structure was observed in the control group than dasatinib group through-transmission electron microscope. After 1000 cells were inoculated in 6-well plates, number of clones was 447 ± 69 in control group,527±39 in dasatinib group.(?) Contrasting miRs expression profiles of megakaryocytes treated with dasatinib and without, incubated with ITP plasma and with normal AB-type plasma, we found the difference of 8 kinds of has-miRs was more than 2.5 fold by cluster analysis.(?) It was verified that miR-146 was correlated with ITP progression closely by detecting plasma of 37 ITP patients through QRT-PCR.Conclusion(?) Dasatinib enhances megakaryocyte proliferation, inhibits platelet production, and up-regulates miR-146 expression.(?) MiR-146 is correlated to the prognosis of ITP closely. |
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