Objective To explore the induction of cardiomyogenesis of microRNA-129 （mir-129） in rat bone marrowmesenchymal stem cells （BM-MSCs） and its mechanism. Methods BM-MSCs were isolated from Sprague-Dawley rats and cultured in vitro. Overexpression of mir-129 or both mir-129 and glycogen synthase kinase-3β （GSK-3β） in BM-MSCs was produced with a lentiviral vector system. All the BM-MSCs were divided into four groups: control group （MSCs），Lentiviral vectors＋MSCs group （Lv-MSCs），mir-129 transfection group （mir-129-MSCs），and mir-129＋GSK-3βdouble transfection group （mir-129＋GSK-3β-MSCs）. Five-Azacytidine （5-Aza） （10 μmol/L） was used to induce BM-MSCsdifferentiation into cardiomyocytes. On the 1st，5 th，10 th，15 th and 20 th day after induction，realtime-PCR was performedto detect mRNA levels of GATA-4，Nkx2.5 and MEF-2C. On the 10 th，15 th and 20 th day after induction，Western blottingwas performed to examine expression levels of cTnI，Desmin，GSK-3β，phosphorylated β-catenin and dephosphorylated β-catenin. Results Compared with the control group，at respective time points，mRNA levels of cardiomyogenic genes and expression levels of cardiomyocyte-related proteins of mir-129 transfection group were significantly elevated，theexpression level of GSK-3β was significantly decreased，and the ratio of dephosphorylated/phosphorylated β-catenin was significantly elevated. When both mir-129 and GSK-3β were overexpressed in BM-MSCs，mRNA levels of cardiomyogenicgenes and expression levels of cardiomyocyte-related proteins were significantly lower than those of mir-129 transfection group，and the ratio of dephosphorylated/phosphorylated β-catenin was significantly decreased. Conclusion Overexpression of mir-129 can promote cardiomyogenesis of rat BM-MSCs possibly via inhibiting GSK-3β production and thus decreasing the inhibition of phosphorylation of β-catenin which then enters the nucleus and activates downstream signaling pathways that regulate cardiomyogenic differentiation of BM-MSCs.
Object ive To summa r i z e the advanc ement of cytoske l e ton and axon outgrowth of neuron. Methods The recent l iterature concerning cytoskeleton and axon outgrowth of neuron was reviewed and summarized. Results The actin filaments and microtubules in neuron were highly polarized and dynamic structures confined to the ti ps of axons and the reci procal interactions between these two major cytoskeletal polymers was also dynamic. Attractive or a repulsive cue whose final common path of action was the growth cone cytoskeleton mediated the growth of axons of neuron by intracellular signaling cascades. Regulating the actin filament and microtubule dynamics as well as their interactions in growth cones played a key role in neurite outgrowth and axon guidance. Rho-GTPases and glycogen synthase kinase 3β (GSK-3β), the two major intracellular signal ing pathways had emerged in recent years as candidates for regulating the dynamics of actin filaments and microtubules. Conclusion The axon outgrowth and guidance depend on well-coordinated cytoskeletal and reciprocal interaction dynamics which also mediate axon regeneration after spinal cord injury. Regulating activity of Rho-GTPases and GSK- 3β simultaneously may acts as key role to regulate the dynamics of cytoskeletal and to determine axon outgrowth.
ObjectiveTo study the effects of Ghrelin for glucose metabolism and insulin sensitivity of L6 rat myoblasts in palmitic acid induced, and to explore its possible mechanisms. MethodsThe L6 rat myoblasts were cultured until differentiation, then using palmitic acid(0.3 mmol/L) for 16 hours. The experimental group was treated with different doses of Ghrelin(1, 10, and 100 nmol/L) for 8 hours, then the glucose uptake was detected by using glucose oxidase peroxidase method(GOD-POD), the cell membrane glucose transporter 4(GLUT-4) protein staining was observated under confocal microscopy, and the expressions of total protein kinase B(Akt), phosphorylated protein kinase B(pAkt), total glycogen synthase kinase-3β(GSK-3β), and phosphorylated glycogen synthase kinase-3β(pGSK-3β) were detected by using immunoblotting(Western blot). ResultsGhrelin enhanced the glucose uptake of L6 rat myoblasts with insulin resistance, the cell membrane Glut-4 stain was deepen, the expressions of pAkt and pGSK-3βprotein increased, and this effect could be PI3K blocker(LY294002) eliminated. ConclusionGhrelin promotes the glucose uptake of L6 rat myoblasts through PI3K/Akt/GSK-3βsignaling pathway, so as to improve the sensitivity of insulin in L6 rats muscle cells.