RESUMO
<p><b>OBJECTIVE</b>To construct an eukaryotic expression vector for miRNA-1-2 that can be expressed in rat H9C2 cardiomyocytes.</p><p><b>METHODS</b>The precursor miRNA (pre-miRNA) DNA template for miRNA-1-2 was designed and generated by PCR amplification. The DNA template was inserted into the hairpin RNA expression vector pSilence-4.1-neo and identified by DNA sequencing analysis. The recombinant plasmid DNA was then transfected into H9C2 cells via Lipofectamine, and the green fluorescence protein expression vector pEGFP-N3 served as the transfection marker. Twenty-four hours after transfection, the total cellular RNA was extracted using TRIzol reagent, and thermoscript reverse transcriptase (RT)-PCR was performed to determine miRNA-1-2 precursor expression.</p><p><b>RESULTS AND CONCLUSION</b>DNA sequencing indicated that the miR-1-2 expression plasmid was correctly constructed. The precursor miRNA-1-2 was successfully expressed in the H9C2 cells, and the expression of Hand2 protein could be efficiently inhibited by miRNA-1.</p>
Assuntos
Animais , Ratos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Genética , Metabolismo , Linhagem Celular , Regulação para Baixo , Proteínas de Fluorescência Verde , Genética , Metabolismo , MicroRNAs , Genética , Miócitos Cardíacos , Biologia Celular , Metabolismo , Plasmídeos , Genética , RNA Mensageiro , Genética , RNA Interferente Pequeno , Genética , TransfecçãoRESUMO
<p><b>OBJECTIVE</b>To investigate the effect of adenoviral vector infection on the differentiation potential of human bone marrow mesenchymal stem cells (hMSCs).</p><p><b>METHODS</b>The third-passage hMSCs were infected with the recombinant adenovirus expressing green fluorescence protein (GFP) for 2, 4, 8 and 16 days. RT-PCR was used to detect the mRNA expression of endodermal marker CYP 51, mesodermal marker SM22alpha, ectodermal marker nestin, pluripotent marker oct-4 and the alternative splicing factor nPTB. Seven days after adenovirus infection, the hMSCs were cultured in the presence of adipogenic agents for 14 days, and the adipose cells differentiated from hMSCs were detected with oil red O staining.</p><p><b>RESULTS</b>Compared with normal hMSCs, the cell infected with the adenovirus for 2, 4, 8 and 16 days showed no obvious down-regulation of CYP51, SM22alpha, nestin, OCT4 or nPTB. The hMSCs 7 days after adenovirus infection were induced to differentiate into adipose cells, with a similar differentiation rate to that of normal hMSCs. CONCLUSION The differentiation potential of hMSCs is not affected by adenovirus infection, suggesting that adenovirus can be used as the gene delivery vector in MSC differentiation studies.</p>
Assuntos
Humanos , Adenoviridae , Genética , Fisiologia , Células da Medula Óssea , Biologia Celular , Diferenciação Celular , Fisiologia , Células Cultivadas , Vetores Genéticos , Genética , Proteínas de Fluorescência Verde , Genética , Interações Hospedeiro-Patógeno , Células-Tronco Mesenquimais , Biologia Celular , Metabolismo , RNA Mensageiro , Genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
@#ObjectiveTo observe the curative effect of marrow stem cell transplant on bone nonunion and bone defection.Methods140 patients were randomly divided into the group A (with auto-iliac bone transplant) and group B (with auto-marrow stem cell transplant). There were 70 cases in each group. All patients in two groups were treated by operation and inside and outside fixation.ResultsAfter treatment, the average cicatrization time of group A was (7.0±2.0) months, that of group B was (5.0±1.5) months, there was a significant difference between two groups( P<0.05). There were no obvious adverse reactions found during the treatment period.ConclusionCompared with the traditional bone grafting, treating bone nonunion and bone defection by auto-marrow stem cell transplant has obvious superiority with better curative effect, short course and no adverse reactions.