Volume 7 Supplement 1
Retroviral vector-mediated hypoxia-regulated neurotrophin-3 gene transfer reduces apoptosis induced by hypoxia in PC12 cells
© Zhang et al; licensee BioMed Central Ltd. 2012
Published: 7 February 2012
Gene therapy for ischemic diseases is a prospective strategy. However, excessive expression of therapeutic genes may produce undesired side effects. Recently, multiple copies hypoxia response elements (HRE) were developed to conditionally regulate gene expression under hypoxia. As a nerve growth factor, Neurotrophin-3 (NT-3) possesses neural protect effects either in vitro or in vivo. To explore hypoxia-controlled NT-3 expression, we constructed a recombinant retrovirus vector with 5HRE and NT-3, and generated a gene transferred cell line PC12-5HRE-NT3 to determine effects of conditionally expressed NT-3 on apoptosis induced by hypoxia in PC12 cells.
Five copies of HRE from human VEGF gene and simian virus 40 minimal promoter (SV40mp) were employed to construct a cassette and neurotrophin-3 was inserted into its downstream to generate RV-5HRE-NT3. Mediated by retrovirus, 5HRE-NT3 was transferred into PC12 cells and screened with G418, gene transferred cell lines were generated and identified by reporter gene EGFP, Reverse transcription PCR (RT-PCR) and immunofluorescence cell staining. Conditional expression of NT-3 was detected by RT-PCR, ELISA and Western blot. Apoptosis induced by hypoxia was evaluated by TUNEL. Apoptosis related molecules, phosphorylated p38 and active Caspase-3 were assayed by Western blot.
We successfully constructed the retroviral vector RV-5HRE-NT3 and generated the gene transferred cell line PC12-5HRE-NT3. Under normoxia, NT-3 expressed at an extremely low level in PC12-5HRE-NT3, whereas it significantly increased under hypoxia (P < 0.05). There was no significant difference in PC12-NT3 between normoxia and hypoxia. Conditionally expressed NT-3 reduced apoptosis induced by hypoxia in gene transferred cell line PC12-5HRE-NT3(P < 0.05) but not PC12-5HRE-EGFP or PC12 cells. Additionally, activation of p38 and Caspase-3 were depressed in PC12-5HRE-NT3 under hypoxia, which indicated that both of them were involved in the protective effect against apoptosis.
These results suggest that 5HRE-SV40mp is an ideal cassette to regulate therapeutic gene expression in response to hypoxia, and 5HRE-NT3 can be further developed as an effective therapeutic gene to provide neuroprotection against cerebral ischemia.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.