RESUMEN
Objective: In this study, we describe an efficient approach for stable knockdown of adenosine kinase [ADK] using lentiviral system, in an astrocytoma cell line and in human Wharton's jelly mesenchymal stem cells [hWJMSCs]. These sources of stem cells besides having multilineage differentiation potential and immunomodulatory activities, are easily available in unlimited numbers, do not raise ethical concerns and are attractive for gene manipulation and cell-based gene therapy
Materials and Methods: In this experimental study, we targeted adenosine kinase mRNA at 3' and performed coding sequences using eight miR-based expressing cassettes of anti-ADK short hairpin RNA [shRNAs]. First, these cassettes with scrambled control sequences were cloned into expressing lentiviral pGIPZ vector. Quantitative real time-polymerase chain reaction [qRT-PCR] was used to screen multi-cassettes anti-ADK miR-shRNAs in stably transduced U-251 MG cell line and measuring ADK gene expression at mRNA level. Extracted WJMSCs were characterized using flow cytometry for expressing mesenchymal specific marker [CD44+] and lack of expression of hematopoietic lineage marker [CD45-]. Then, the lentiviral vector that expressed the most efficient anti-ADK miR-shRNA, was employed to stably transduce WJMSCs
Results: Transfection of anti-ADK miR-shRNAs in HEK293T cells using CaPO4 method showed high efficiency. We successfully transduced U-251 cell line by recombinant lentiviruses and screened eight cassettes of anti-ADK miR- shRNAs in stably transduced U-251 MG cell line by qRT-PCR. RNAi-mediated down-regulation of ADK by lentiviral system indicated up to 95% down-regulation of ADK. Following lentiviral transduction of WJMSCs with anti-ADK miR- shRNA expression cassette, we also implicated, down-regulation of ADK up to 95% by qRT-PCR and confirmed it by western blot analysis at the protein level
Conclusion: Our findings indicate efficient usage of shRNA cassette for ADK knockdown. Engineered WJMSCs with genome editing methods like CRISPR/cas9 or more safe viral systems such as adeno-associated vectors [AAV] might be an attractive source in cell-based gene therapy and may have therapeutic potential for epilepsy
RESUMEN
Introduction: Transforming Growth Factor-Beta 1 [TGF-beta1] is a pleiotropic cytokine with potent anti-inflammatory property, which has been considered as an essential risk factor in the inflammatory process of Ischemic Stroke [IS], by involving in the pathophysiological progression of hypertension, atherosclerosis, and lipid metabolisms. -509C/T TGF-beta1 gene polymorphism has been found to be associated with the risk of IS. The aim of this meta-analysis was to provide a relatively comprehensive account of the relation between -509C/T gene polymorphisms of TGF-beta1 and susceptibility to IS
Methods: Male Wistar rats were divided into sham [receiving phosphate buffered saline within dorsal hippocampus], pilocarpine [epileptic model of TLE], single injection BDNF [epileptic rats which received single high dose of BDBF within dorsal hippocampus], and multiple injections BDNF [epileptic rats which received BDNF in days 10, 11, 12, and 13 after induction of TLE] groups. Their electrocorticogram was recorded and amplitude, frequency, and duration of spikes were evaluated
Results: Amplitude and frequency of epileptiform burst discharges were significantly decreased in animals treated with BDNF compared to pilocarpine group
Conclusion: Our findings suggested that BDNF may modulate the epileptic activity in the animal model of TLE. In addition, it may have therapeutic effect for epilepsy. More studies are necessary to clarify the exact mechanisms of BDNF effects