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To investigate the effect of 27nt-miRNA on the differentiation of mesenchymal stem cells into vascular smooth muscle cells. The highly expression plasmids of 27nt-miRNA and anti-27nt-miRNA, and negative control plasmids were constructed, packaged with lentivirus and transfected into human umbilical cord mesenchymal stem cells (hUCMSCs). Collagen IV was added to induce hUCMSCs differentiation into blood vessel smooth muscle cells (VSMCs). The cell viability was measured by MTT assay. The expression of SMA, SM22α at mRNA and protein levels was determined by RT-PCR, immunocytochemical staining and Western blotting. Compared with the negative control group, the viability of the 27nt-miRNA overexpression group was decreased by 20.48% (P<0.05), and the expression of SMA mRNA and SM22α mRNA and protein was significantly increased (P<0.05); the viability of Anti-27nt-miRNA group was increased 18.07% (P<0.05), and the expression of SMA mRNA and SM22α mRNA and protein was decreased (P<0.05). In summary, 27nt-miRNA promotes mesenchymal stem cells differentiation into vascular smooth muscle cells and inhibits cells viability.
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Humans , Cell Differentiation , Cells, Cultured , Mesenchymal Stem Cells , MicroRNAs , Muscle, Smooth, Vascular , Myocytes, Smooth MuscleABSTRACT
Objective To explore the monitoring of cerebrospinal fluid (CSF) dynamics in a model of brain herniation induced by acute intracranial hypertension in Guangxi Bama-Mini pigs by phasecontrast cine magnetic resonance imaging (PC cine MRI).Methods Femoral artery blood were extracted from 10 pigs,and injected into the frontal and temporal parietal lobe to make a model of brain herniation induced by acute intracranial hypertension.The mean arterial blood pressure (MAP),intracranial pressure (ICP),and cerebral perfusion pressure (CPP) were monitored.Routine T1WI,T2WI,coronal,sagittal and cerebrospinal fluid flow sequence (fast PC cine slice) which positioned on the cervical 3 (C3) vertebral body as the center and perpendicular to the spinal scans were performed on all experimental animals before and after blood injection with 3.0T Magnetic Resonance Imaging.The ICP,MAP,CPP,the absolute values of CSF peak flow velocity and the absolute value of carotid peak flow velocity before and after blood injection were compared.Results The ICP,MAP,CPP,and the absolute value of CSF peak flow velocity before injection of autologous arterial blood were statistically significant as compared with those after blood injection [(6.80±2.044) mmHg vs (52.20±1.619) mmHg,(76.80±7.068) mmHg vs (142.80±12.399) mmHg,(70.00±6.074) mmHg vs (90.50±12.250) mmHg,and the absolute value of CSF peak flow velocity was (243.20±77.671) mm/s vs (201.40±55.482) mm/s,respectively,P＜0.01].The absolute value of the peak velocity of the carotid artery before blood injection was not statistically significant compared with that after blood injection [(876.80±239.908) mm/s vs (799.40±241.829) mm/s,P＞0.05].Conclusion After the formation of brain herniation induced by acute intracranial hypertension,the CSF flow in the C3 level spinal canal showed a low dynamic change,and the CSF flow velocity waveform was disordered and malformed.The non-invasive measurement of CSF dynamics by PC cine MRI can provide an important basis for the change of CSF dynamics in the model of brain herniation induced by acute intracranial hypertension,and provide a theoretical basis for further research on damage control neurosurgery in the future.
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Objective To investigate the differences in eNOS gene expression,activity and its metabolites before and after human bone marrow mesenchymal stem cells (hBMSCs) are induced into vascular endothelial cells.Methods hBMSCs were induced into vascular endothelial cells.The morphological changes of the cells were observed under inverted microscope.Transwell assay was used to detect the cells' migration ability.The protein expression of eNOS was detected by immunofluorescence and Western blot.The activity of eNOS was detected by ELISA and the content of NO in cell culture supernatant was determined by nitrate reduction method.Results Compared with those in the undifferentiated group,the morphological changes of the differentiated cells were obvious.Cell migration ability increased by 238.10％ (73.000±7.002 vs.21.000±4.359,P＜0.05).The expression of eNOS protein increased by 114.72％ (0.423±0.011 vs.0.197±0.079,P＜0.05).The activity of eNOS was enhanced by 157.49％ (4.967±0.073 vs.1.929±±0.103,P＜0.05).The synthesis and release of NO increased by 155.67％ (184.909±1.853 vs.72.323±0.426,P＜0.05).Conclusion After hBMSCs are induced into endothelial cells,the expression of eNOS gene increases,their activities increase,synthesis and release of the metabolite NO increase.It may provide a basis for prevention and treatment of cardiovascular diseases with stem cells.
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Objective To further investigate the molecular mechanism of microRNA(miRNA)-24 on endothelial nitric oxide synthase(eNOS)gene expression,activity and the impact on its metabolites. Methods The plasmid of miRNA-24 was constructed and transfected to human umhilical vein endothelial cells(HUVECs). The proliferation of cells was detected by MTT test. Migration ability was measured by scratch wound model. The mRNA transcription and protein expressions of eNOS were determined by RT-PCR and Western blot respectively. The eNOS activity was detected by ELISA. The metabolite NO production in cell culture supernatant was detected by nitrate reduction. Results Compared with control group,the proliferation of endothelial cells in the miRNA-24 group was decreased by 54.32%;the migration of HUVECs was reduced by 48.62%;the expressions of eNOS mRNA and protein were de?creased by 43.92%and 42.71%,respectively. miRNA-24 suppressed eNOS activity by 73.20%. Meanwhile,the NO production was decreased by 55.29%. Conclusion miRNA-24 inhibits the synthesis and release of metabolite NO,which may be a molecular target of prevention and treatment in cardiovascular diseases.
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Objective: To investigate the effects of miR-24 on endothelial nitric oxide synthase (eNOS) gene expression with regulation and endothelial cell proliferation, migration, tube formation in human umbilical vein endothelial cells (HUVECs). Methods: Constructed high expression plasmid of miR-24 and miR-24 antisense sequence were introduced into HUVECs and the cells included in 3 groups: Control group, miR-24 group and miR-24 inhibitor group. HUVEC proliferation was detected by MTT test, migration was measured by Scratching and Transwell methods, tube formation was examined by Matrigel assay; mRNA and protein expressions of eNOS and Sp1were determined by RT-PCR and Western blot analysis respectively. Results:①Compared with Control group, miR-24 group had decreased cell proliferation by 45.45% as (0.36 ± 0.04) vs (0.66 ± 0.08),P<0.05; miR-24 group had lower speed of cell migration, decreased number of cell migration by 74.75% as (30.25±3.78) vs (119.80±10.94),P<0.01 and there was no obvious tube formation.②Compared with Control group, miR-24 group showed reduced eNOS mRNA expression by 46.2% as (0.49±0.02) vs (0.91±0.01),P<0.05, reduced protein expression by 49.07% as (0.55±0.05) vs (1.08±0.05),P<0.05; meanwhile, decreased Sp1 mRNA expression by 44.9% as (0.49±0. 01) vs (0. 89±0.02)P<0.05, decreased protein expression by 54.90% as (0.46±0.02) vs (1.02±0.04),P<0.05. In miR-24 inhibitor group, the above indexes were lower than Control group but higher than miR-24 group, the amount of tube formation and the length of tubes were similar between Control group and miR-24 inhibitor group. Conclusion: MiR-24 may inhibit HUVECs proliferation, migration, tube formation and suppress eNOS expression; Sp1 might be one of the important regulators.
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[ABSTRACT]AIM:ToinvestigatewhethermiRNA-24isinvolvedintheregulationofendothelialnitricoxide synthase ( eNOS ) expression and vascular endothelial cell proliferation .METHODS: A plasmid that highly expressed miRNA-24 was constructed, and was transfected into the human umbilical vein endothelial cells (HUVECs) by liposome. The cell proliferation was detected by MTT assay .The expression of eNOS and Sp 1 at mRNA and protein levels was exa-mined by real-time PCR, immunohistochemistry and Western blotting .RESULTS:Compared with control group , the pro-liferation of endothelial cells in miRNA-24 group was significantly decreased by 41.97 % (0.47 ±0.04 vs 0.81 ±0.03, P<0.01), and the expression of eNOS at mRNA and protein levels was decreased by 44.8% (0.48 ±0.01 vs 0.87 ± 0.03, P<0.05) and 71.92%(0.16 ±0.06 vs 0.57 ±0.08, P<0.05), respectively.Meanwhile, the mRNA and pro-tein levels of Sp1 were significantly decreased by 53.00% (0.45 ±0.02 vs 0.93 ±0.01, P<0.05) and by 62.31%(0.13 ±0.07 vs 0.31 ±0.09, P<0.05), respectively.In miRNA-24 inhibitor group, the above indexes were decreased compared with control group , but significantly increased compared with miRNA-24 group.CONCLUSION: miRNA-24 significantly inhibits the proliferation of HUVECs and the eNOS expression .Sp1 possibly acts as one of the important factors in the regulation of eNOS expression by miRNA-24.
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Aim To explore the effect of microRNAs on the differentiation of 3T3-L1 adipocytes and the expression of adipo-related gene-fatty acid binding protein during the adipocyte differentiation.Methods adipo-related microRNAs during 3T3-L1 adipocyte differentiation were screened and identified by micorRNA microarray.Constructed high-expression plasmids of the adipo-related microRNAs,were transfected into the 3T3-L1 preadipocytes by lipofectamine.While the effect of adipo-related microRNAs on the course of 3T3-L1 adipocyte differentiation was observed,the protein and mRNA expression level of fatty acid binding protein(FABP4)were analyzed by Western blot and RT-PCR during 3T3-L1 adipocyte differentiation.Results The expression profiles of microRNAs have significant changed during 3T3-L1 adipocyte differentiation,in which 35 microRNAs among them down-relation,the most lowly expression is miR-24;17 microRNAs among them up-relation,the most highly expression is miR-21.MiR-24 significantly inhibited adipocyte differentiation and maturity,while miR-21 have no significant effect.MiR-24 significantly inhibited the expression of FABP4,but had no effect on the level of its mRNA;miR-21 had no effect on the expression of protein and mRNA of FABP4.Conclusion There exist adipogenic-related microRNAs during 3T3-L1 adipocyte differentiation; miR-24 play an important role in the regulation of 3T3-L1 preadipocyte differentiation into adipocyte and the(FABP4)protein expression.