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Cell Journal، جلد ۲۰، شماره ۱، صفحات ۱-۹
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عنوان انگلیسی Stable Knockdown of Adenosine Kinase by Lentiviral Anti-ADK miR-shRNAs in Wharton’s Jelly Stem Cells
چکیده انگلیسی مقاله Objective: Astrogliosis is a pathological hallmark of neurological diseases like epilepsy, traumatic brain injury (TBI), Alzheimer’s disease and autism. Studies have shown the relation between overexpression of Adenosine kinase (ADK) and astrogliosis in molecular investigations. RNA interference (RNAi) technology is a useful and promising tool for downregulation of adenosine kinase for compensating neural dysfunction created by adenosine kinase overexpression. In this study, we describe an efficient approach for stable knockdown of adenosine kinase by the lentiviral system, first in an astrocytoma cell line and then in human Wharton’s jelly mesenchymal stem cells (hWJMSCs). These source of stem cells besides to have multilineage differentiation potential and immunomodulatory features are more feasible to access in an unlimited number without concerning about ethical and technical issues, attractive for gene manipulation and cell-based gene therapy. In this study, we improved therapeutic potential of WJMSCs as adenosine-releasing stem cells by stable knockdown of ADK. Materials and methods: In this experimental study, we targeted adenosine kinase mRNA at 3' and coding sequences by eight miR-based expressing cassettes of anti-ADK shRNAs. First, these cassettes with scrambled control sequence were cloned into expressing lentiviral pGIPZ vector. Quantitative real- time PCR (qRT-PCR) used to screen multi-cassettes anti-ADK miR-shRNAs in stably transduced U-251 MG cell line and measuring ADK gene at mRNA level. Extracted WJMSCs were characterized with flow cytometry for expressing mesenchymal specific marker (CD44+) and lack of hematopoietic lineage marker (CD45-). Then, the lentiviral vector that expresses the most efficient anti-ADK miR-shRNA was employed to stable transduction of WJMSCs. Results: Transfection of anti-ADK miR-shRNAs in HEK293T cells by Ca-Po4 method showed high efficiency. We transduced U-251 cell line successfully by recombinant lentiviruses. We screened eight cassettes of anti-ADK miR-shRNAs in stable transduced U-251 MG cell line by qRT PCR. RNAi-mediated downregulation of adenosine kinase by lentiviral system indicated up to 95% downregulation of adenosine kinase. Following lentiviral transduction of WJMSCs with anti-ADK miR-shRNA expression cassette, we implicated also, downregulation of ADK up to %95 by qRT-PCR and confirmed it by western blot at the protein level. Conclusion: Our findings indicate efficient shRNA cassette for knockdown of adenosine kinase. Engineered WJMSC with genome editing methods like CRISPR/cas9 or more safe viral system such as adeno associated vector (AAV) might be an attractive source in cell-based gene therapy and may have a therapeutic potential in epilepsy.
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نویسندگان مقاله | Hajar Estiri


| Ali Fallah


| Masoud Soleimani


| Abbas Aliaghaei


| Fariba Karimzadeh


| Shahnaz Babaei Abraki


| Mohammad Hossein Ghahremani


نشانی اینترنتی http://celljournal.org/journal/article/abstract/4916
فایل مقاله اشکال در دسترسی به فایل - ./files/site1/rds_journals/16/article-16-851794.pdf
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