Document Type : Original Article
Authors
1
Science and Research Branch, Islamic Azad University, Tehran, Iran
2
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran;Mehr Infertility Research Center, Rasht, Iran;4Department of Cell Biology and Anatomical Science, Faculty of Medicine, Sh
3
5Systems and Synthetic Biology Group, Mede Bioeconomy Company, Tehran, Iran
4
6Department of Molecular Biology and Genetic Engineering, Stem Cell Technology Research Center, Tehran, Iran
5
7Department of Biology, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran
6
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran;Mehr Infertility Research Center, Rasht, Iran
7
8Department of Medical Genetics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences and Health Services, Tehran, Iran
8
9Cellular and Molecular Biology Research Center, Babol University of Medical Sciences, Babol, Iran
9
0Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
10
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran;Department of Biotechnology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Abstract
Objective
Autoimmune diseases precede a complex dysregulation of the immune system. T helper17 (Th17) and interleukin (IL)-17 have central roles in initiation of inflammation and subsequent autoimmune diseases. IL-27 significantly controls autoimmune diseases by Th17 and IL-17 suppression. In the present study we have created genetic engineered mesenchymal stem cells (MSCs) that mediate with lentiviral vectors to release IL-27 as an adequate vehicle for ex vivo gene therapy in the reduction of inflammation and autoimmune diseases.
Materials and Methods
In this experimental study, we isolated adipose-derived MSCs (AD-MSCs) from lipoaspirate and subsequently characterized them by differentiation. Two subunits of IL-27 (p28 and EBI3) were cloned in a pCDH-513B-1 lentiviral vector. Expressions of p28 and EBI3 (Epstein-Barr virus induced gene 3) were determined by real time polymerase chain reaction (PCR). MSCs were transduced by a pCDH-CMV-p28-IRES- EBI3-EF-copGFP-Pur lentiviral vector and the bioassay of IL-27 was evaluated by IL-10 expression.
Results
Cell differentiation confirmed true isolation of MSCs from lipoaspirate. Restriction enzyme digestion and sequencing verified successful cloning of both p28 and EBI3 in the pCDH-513B-1 lentiviral vector. Real time PCR showed high expressions level of IL-27 and IL-10 as well as accurate activity of IL-27.
Conclusion
The results showed transduction of functional IL-27 to AD-MSCs by means of a lentiviral vector. The lentiviral vector did not impact MSC characteristics.
Keywords
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