ObjectiveTo study microRNA (miRNA) involved in the regulation of sinus cell differentiation by comparing sinus node, atrial myocardium, and ventricular myocardium specific miRNA expression profile differences in Kunming mice. MethodsA total of 180 Kunming mice, aged 60-90 days and weighing 35-45 g, were selected without gender differences after the method of anatomical localization for sinus node had been confirmed by preliminary experiments in another 10 Kunming mice. All the sinus node, atrial myocardium, and ventricular myocardium tissue from 180 mice were dissected and frozen by liquid nitrogen. The structure of tissue was observed by HE staining. Total RNA were extracted and quality-controlled before hybridize with miRNA chip. The chips with miRNA were used to screen specific miRNAs; and correlation analysis of gene function was done. ResultsThe area of mice sinus node located at juncture of the superior vena cava and the right atrium junction with crista as its longitudinal axis, ranged 2.0 mm×1.5 mm×1.0 mm. HE staining showed the sinus cells were less, with no stripes, lightly stained cytoplasm, large and round nucleus, and there were much fibrous connective tissue around cells with a visible sinus node artery. The miRNA microarray results showed that compared with atrial myocardium and ventricular myocardium, there were 39 differentially expressed miRNAs in sinus node, including 12 up-regulated miRNAs and 27 down-regulated miRNAs. Based on the regulatory networks of differential miRNA and target gene, the regulatory miRNA was obtained. ConclusionThe differentially expressed miRNA in mice sinus node possibly may be involved in the regulation of sinus cell differentiation.
ObjectiveTo investigate whether Akt1 gene transfection mediated by recombinant lentivirus (LVs) in the bone marrow mesenchymal stem cells (BMSCs) could enhance the ability of hypoxia tolerance so as to provide a theoretical basis for improving the effectiveness of stem cells transplantation. MethodLVs was used as transfection vector, enhanced green fluorescent protein (EGFP) was used as markers to construct the pLVX-EGFP-3FLAG virus vector carrying the Akt1 gene. The 3rd generation BMSCs from 3-5 weeks old Sprague Dawley rats were transfected with pLVX-EGFP virus solution as group B and with pLVX-EGFP-3FLAG virus solution as group C; and untransfected BMSCs served as control group (group A). At 2-3 days after transfection, the expression of green fluorescent was observed by fluorescence microscope; and at 48 hours after transfection, Western blot method was used to detect the expression of Akt1 protein in groups B and C. BMSCs of groups B and C were given hypoxia intervention with 94%N2, 1%O2, and 5%CO2 for 0, 3, 6, 9, and 12 hours (group B1 and group C1) . The flow cytometry was used to analyze the cell apoptosis rate and cell death rate, and the MTT method to analyze the cell proliferation, and Western blot to detect the expression of apoptosis related gene Caspase-3. ResultsAfter transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in groups B and C, the transfection efficiency was about 60%. Akt1 expression of group C was significantly higher than that of group B (t=17.525, P=0.013) . The apoptosis rate and cell death rate of group B1 increased gradually with time, and difference was significant (P<0.05) . In group C1, the apoptosis rate and cell death rate decreased temporarily at 3 hours after hypoxia intervention, then increased gradually, and difference was significant (P<0.05) . The apoptosis rate and cell death rate of group C1 were significantly lower than those of group B1 at each time point (P<0.05) except at 0 hour. MTT assay showed that absorbance (A) values of groups B and C were significantly higher than those of groups B1 and C1 at each time point (P<0.05) ; the A value of group B was significantly lower than that of group C at each time point (P<0.05) . The A value of group B1 was significantly lower than that of group C1 at 6, 9, and 12 hours after hypoxia intervention (P<0.05) . Western blot results showed that the Caspase-3 expression of group C1 significantly reduced when compared with group B1 at each time point (P<0.05) . ConclusionsAkt1 gene transfection mediated by recombinant LVs could significantly improve hypoxia tolerance of BMSCs by inhibiting the apoptosis, which could provide new ideas for improving the effectiveness of stem cells transplantation.