RESUMO
Objective To investigate the expression and role of Huntingtin-associated protein-1 (HAP-1) in the process of valproate acid (VP A) inducing neural stem cells (NSCs) into neurons. Methods The hippocampus NSCs of SD rats were isolated and cultured, Real-time PCR and Western blotting were used to detect HAP-1 mRNA and protein expression at day 0, day 1, day 3 and day 5 during the induction of VPA on NSCs differentiation into neurons ; Real-time PCR was used to detect the expression level of HAP-1 mRNA in multiple tissues of adult SD rats, as well as NSCs, neurons and astrocytes. After applying small interfering RNA technology to down-regulate the expression of HAP-1 mRNA in NSCs, Real-time PCR was used to detect the mRNA expression levels of neuron-specific molecules stathmin-2 ( Stmn-2), neuronal differentiation-1 (Neurod-1), microtubule-associated protein-2 (Map-2) and synapsin-1 (Syn-1), and Western blotting was used to detect the protein expression levels of neuron-specific marker β-tubulin III (Tuj-1). Immunofluorescence was used to detect the proportion of NSCs differentiated into Tuj-1 positive neurons, and to observe the development of neurons. Results At day 1 and day 3 after VPA treatment, the expression of HAP-1 mRNA and protein in the VPA group was significantly up-regulated; HAP-1 mRNA was predominantly expressed in the hippocampus, and its expression was higher in neurons, followed by NSCs, and minimally in astrocytes. After down-regulating HAP-1 with small interference technology, the proportion of NSCs differentiated into Tuj-1 positive neurons reduced, and neuron development became worse. Conclusion VPA may promote the differentiation of NSCs into neurons by up-regulating HAP-1 expression.
RESUMO
To establish a UFLC-MS/MS method for the determination of valproate acid in human plasma. Methods:The sample was precipitated by methanol. The analysis of valproate acid and diclofenac sodium ( internal standard) was carried out on a Shim-pack XR-ODS II C18 column (2. 0 × 75 mm,2. 2 μm). Gradient elution was adopted with acetonitrile and water (containing 5 mmol·L-1 ammonium acetate) at a flow rate of 0. 3 ml·min-1. The detection was performed with multiple reactions monitoring (MRM) using nega-tive electrospray ionization (ESI) at m/z 142. 8→142. 8 for valproate acid and m/z 294. 0→249. 8 for diclofenac sodium. Results: The calibration curve of valproate acid was linear over the range of 8.4-168.0 μg·ml-1(r=0.997 5). Inter- and intra-day RSDs were less than 15%, and the analysis was proven to be stable. Totally 30 samples determined by EMIT were assayed by the method. And the results of the two methods analyzed by independent t-test showed no statistical significance. Conclusion:The method is rapid,sensitive and spe-cific,which can be applied in the determination of valproate acid in human plasma.