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Abstract

PRODUCTION OF MOUSE EMBRYONIC STEM CELLS EXPRESSING THE GPX1 AND NURR1 GENES BY USING LENTI VIRUS VECTORS; NEW CELLS SOURCES FOR CELL THERAPY IN PARKINSON'S DISEASE

Sajjad Biglari, Mojtaba Safi*, Navid Pourzardosht, Mohammad Ebadi, MD, Saeid Ebadi, MD

ABSTRACT

Background: Parkinson's disease is the second most common neurological developmental disease which has involved the high population of elderly people in our society. In this disease, the dopaminergic neurons (Dan) of the Nigrostriatal are selectively lost. Now the main cause of Parkinson's disease is unknown, but oxidative stress play an important role in the development and progression of disease. Cell therapy as a treatment for the replacement of lost neurons can be a permanent and effective treatment for this disease. Methods: in the present study, by using the techniques of gene transfer based on lenti virus vectors, the Nurr1 genes (a transcription factor involved in the development of Dan) and GPX1 (an antioxidant enzyme) was transferred to the R1 mouse embryonic stem cells. And the permanent expression of these genes was confirmed in these cells. The above transgenic stem cells were differentiated according to a five-step protocol and by treatment of growth factors and molecules on Matrigel with Poly-L-Lysine. and at the each of differentiation stage, expressing specific markers of each stage were studied by using the real time PCR techniques and immunocytochemistry techniques. Results: the results show that the infected stem cells with lenti virus, after receiving GPX1 and Nurr1 genes, still have maintained their pluripotency characteristics. Also, in infected cells, the transgenes are expressed at the mRNA level and the ICC results showed that the Nurr1 protein is expressed in the nucleus of infected cells and the GPX1 protein is expressed in the cytoplasm of these cells. Investigating the pluripotency markers expressing, Nanog protein in these cells as the RT-PCR shows for this market that the expression of pluripotent stem cell markers in these cells are at the acceptable level. Conclusions: the results of this study can be used in preclinical cell therapy of Parkinson's disease and transplantation to the animal model. Also, cell therapy as a treatment for the replacement of lost neurons can be a permanent and effective treatment for this disease.

Keywords: mouse embryonic stem cells differentiation, Nurr1, GPX1, lenti virus vectors, Parkinson.


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