Cell and Tissue Journal

Abstract -
Aim: In this study Nurr1 and GDNF, due to their anti-inflammatory and regenerative effects and Nurr1-mediated regulation of GDNF receptor (Ret) expression, were selected to protect dopaminergic SH-SY5Y cell line against neuroinflammation and toxicity caused by 6-OHDA.
Material and Methods: Recombinant lentiviral vectors carrying Nurr1 and GDNF genes were prepared and transdused to SH-SY5Y and astrocytoma (1321N1) cell lines respectively. Also HEK-293T cells were transfected with plasmid carrying GDNF to overexpress this factor; condition media of transduced astrocytoma and transfected HEK-293T cells were collected and stored. Next, overexpression of mentioned factors was demonstrated by RT-PCR. On the other hand, microglial cells were isolated from neonatal rat brains and induced with LPS to produce neuroinflammatory factors; Inducible expression of them was demonstrated by Griess test and RT-PCR. Condition media of microglia was collected and saved. Finally, SH-SY5Y cells overexpressing Nurr1 were treated with condition media of transduced astrocytoma / transfected HEK-293T and then with condition media of LPS-induced microglia or 6-OHDA toxin.
Results: data from MTT assay showed, SH-SY5Y cells overexpressing Nurr1 or pretreated with GDNF are more resistant to toxicity caused by neuroinflammation and 6-OHDA. Also Nurr1 and GDNF have cooperative effects and give more protection to dopaminergic cells.
Conclusion: Nurr1 and GDNF each have protective effects on dopaminergic neural cells against inflammatory factors or 6-OHDA. Also they synergize with each other leading to more protection for dopaminergic neural cells.

Abstract Aim: The purpose of this study is to examine DJ-1effect on increasing survival of dopaminergic (DAergic) cells against parkinsonian toxicity.
Material and Methods: First recombinant lenti-viruses transporters were produced with both DJ-1 and reporter Jred genes and were used to cells infection. To this end, three lentiviruses vectors namely transporter, packaging and envelope were applied for co-transfection of HEK-293T cells as virus-producing cell line. 24 and 48 hours transfected cell media were collected and concentrated till lentivirus stock generation. Following transduction of DAergic PC12 cells with this concentrated virus stock, the infected cells overexpressed DJ-1. After treatment of these transduced cells with the 6-OHDA toxin, their survival rate was measured in comparison with control.
Results: Transfection HEK-293T cells steps and PC12 cells transduction with the virus stock were done successful, because reporter Jred gene expression was observed using fluorescence microscope in both steps. Then DJ-1 overexpression was proofed using RT-PCR method. Next experiments indicated that DJ-1 overexpression causes significant increase in PC12 cells survival against produced toxicity of 6-OHDA. PC12 cell survival percentage that had DJ-1-overexpressing was 30% more than control cells survival percentage and this increasing was statistically significant.
Conclusion: increasing of DJ-1 expression in DAergic PC12 cells significantly increased their resistance and perpetuity against 6-OHDA neurotoxicity.

Abstract Aim: The aim of this study was to generate recombinant lentiviruses carrying Nurr1 to express it in human cells. Material and methods: The IRES-EGFP fragment was isolated from the pIRES2-EGFP vector using restriction enzymes BglII/NotI and made blunt-ended using Klenow. The transfer vector PNL-EGFP/CMV/WPREdU3 was digested with NheI/XhoI and made blunt-ended. Finally, the isolated IRES-EGFP fragment was inserted into this lentivirus vector to generate lentivirus construct (I). The human Nurr1 gene was then isolated from the PCMX-NOT vector using BamHI and XhoI and inserted into construct (I) pre-digested with BamHI and SalI. At this step lentivirus construct (II) as our final transfer construct was generated. In order to generate recombinant lentiviruses, we then transfected the HEK-293T cell line with transfer vector plus packaging and envelope vectors. Cell medium full of virus particles was collected and passed through Amicon filters to produce a concentrated virus stock. The stock was ultimately used for transduction of fresh HEK-293T cells. EGFP expression was shown under fluorescent microscope and Nurr1 expression was analyzed using RT-PCR. Results: Enzymatic tests confirmed the correct cloning of the hNurr1gene into the lentivirus backbone. Observation by fluorescent microscopy showed EGFP expression post-transfection and post-transduction. RT-PCR demonstrated Nurr1 expression at both stages. Conclusion: In this study, lentiviruses carrying the human Nurr1 gene were produced and used for transduction of human cell line HEK-293T. The transduced cells successfully expressed the Nurr1 gene.