Human nerve growth factor-β (hNGF-β) plays a major role in the development and survival of the central and peripheral nervous systems; therefore, it can, in principle, be used as a therapeutic agent for the treatment of neurodegenerative diseases. As was shown, hNGF-β stimulates neuronal differentiation in vitro and regulates the expression of neuron-related genes. In our study, expression of the ATP1B1, Clusterin, MAO-A, and DUSP6 genes was evaluated in hNGF-treated PC12 cells. At first, hNGF-β protein was generated in a pET39b/BL21 (DE3) bacterial expression system, and biological activity of this protein was confirmed by differentiation of cultured PC12 cells. These cells were treated by the addition of ... More
Human nerve growth factor-β (hNGF-β) plays a major role in the development and survival of the central and peripheral nervous systems; therefore, it can, in principle, be used as a therapeutic agent for the treatment of neurodegenerative diseases. As was shown, hNGF-β stimulates neuronal differentiation in vitro and regulates the expression of neuron-related genes. In our study, expression of the ATP1B1, Clusterin, MAO-A, and DUSP6 genes was evaluated in hNGF-treated PC12 cells. At first, hNGF-β protein was generated in a pET39b/BL21 (DE3) bacterial expression system, and biological activity of this protein was confirmed by differentiation of cultured PC12 cells. These cells were treated by the addition of recombinant hNGF-β protein in a final dose of 50 ng/ml diluted in RPMI 1640. Then total RNA was extracted from harvested PC12 cells at day 7 after treatment and used to analyze the expression levels of the above-mentioned genes by real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. It was found that purified hNGF-β protein migrated as a clear band of ~37 kDa in SDS-PAGE. Moreover, differentiation of PC12 cells was observed 7 days after hNGF-β treatment. Expression of the ATP1B1 and DUSP6 genes was upregulated (230 and 193%), respectively after addition of hNGF-β protein. In contrast, the MAO-A and Clusterin genes showed practically no change in their expression, as compared to untreated cells. Generally, our study demonstrated that the treatment of PC12 cells with hNGF-β protein induces neural morphological modifications and differences in the expression of certain neuron-related genes. Changes in the expression of these genes will be applied in an experimental model.
