Assessment of atp1b1 gene expression in nerve growth factor-treated pc12 cells
,1 Ali namvar
,2 Soheil moradifard
,3 Azam bolhassani
1. Iranian Comprehensive Hemophilia Care Center, Tehran, Iran
2. Iranian Comprehensive Hemophilia Care Center, Tehran, Iran
3. Iranian Comprehensive Hemophilia Care Center, Tehran, Iran
4. Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
Nerve growth factor (ngf) contains three subunits such as α, β and γ subunits. the β subunit is responsible for ngf biological activity. ngf-β plays an important role in development, maintenance and survival of the central and peripheral nervous systems; thus it can be used as a therapeutic agent for the treatment of neurodegenerative diseases such as alzheimer’s disease. the studies showed that ngf-β induces neuronal differentiation of pc12 pheochromocytoma cells in vitro and regulates the expression of some neuron-related genes. in this study, we evaluated the atp1b1 gene expression in ngf-treated pc12 cells. the glycoprotein subunit of na+/k+-atpase (b1 subunit) regulates the number of sodium pumps transported to the plasma membrane
At first, the pet39b plasmid was used to generate ngf-β in bl21 (de3) bacterial cells. gene expression was induced by addition of isopropyl thio-β-d-galactoside (iptg). the ngf-β protein was purified by ni-nta agarose and its biological activity was confirmed using the differentiation of pc12 cells upon ngf-β stimulation, in vitro. for this purpose, pc12 cells were plated in 12-well plastic culture dishes and the recombinant ngf-β was added in final dose of 50 ng/ ml diluted in rpmi 1640. finally, total rna was extracted from harvested pc12 cells at day 7 after treatment and used to analyze the expression level of atp1b1 gene by real-time pcr.
Our data showed that the recombinant ngf-β could be expressed in e. coli bl21 strain at 4 h after induction at 37°c. the purified ngf-β protein under native conditions migrated as a clear band of ~ 42 kda in sds-page. moreover, the pc12 differentiation was observed after 7 days upon ngf-β addition. the atp1b1 expression was upregulated 2.3-fold as compared to glyceraldehyde-3-phosphate dehydrogenase (gapdh) as a reference gene, following addition of ngf protein.
In general, our study demonstrated that the treatment of pc12 cells with ngf-β protein induces neural morphological changes leading to the regulation of the neuron-related gene such as atp1b1. the expression profile of other neuron-related genes will be studied in near future and the results of this study will be developed in experimental model.
Pc12 cells, ngf-β, atp1b1, real time pcr