Abstract
Stem cells have become a powerful tool in the treatment of many diseases owing to their regenerative ability and rapid promotion of development in regenerative medicine such as in traumatic brain injury. However, the high level of oxidant micro-environment in lesion region leads to more than 99% cells into death. In this study, we used genetic methods to edit Keap1 gene in mesenchymal stem cells, we and observed their antioxidative ability. First, we disturbed the start codon and the 376th amino acid codon of Keap1 in adipose-derived mesenchymal stem cells (Ad-MSCs) with CRISPR/Cas9, respectively, to release Nrf2 from the binding of Keap1. As a result, Nrf2 was activated and localized into nuclei and regulated cellular anti-oxidation. We observed that the cells lacking Keap1 ATG codon showed obvious nuclear localization of Nrf2. Besides lower expression of Bax-1 and lower content of malondialdehyde (MDA) were detected after H2O2 treatment, we also found higher expression of Bcl-2 in Keap1 ATG codon knock-out cells, whereas a higher expression of PCNA was observed only in the Keap1 376th codon-edited cells, whose Bax-1 expression was lower than that in the control cells. Our study revealed that loss of Keap1 resulted in anti-oxidative ability in Ad-MSCs, suggesting that our strategy can hopefully increase the viability of mesenchymal stem cells after grafting. This study is also a frontier exploration to the application of CRISPR/Cas9 in Ad-MSCs.
Highlights
Many neural degenerative diseases, like Alzheimer’s diseases, Parkinson’s diseases, stroke, epilepsy, and spinal cord injury, have become the greatest threats to the modern public health
After being transferred to the polyvinylidene difluoride (PVDF) membrane with 300 mA for 150 min, the PVDF membrane was blocked in Tris-buffered saline (TBS)−0.05% Tween 20 with 5% non-fat milk in 37◦C for 60 min, and primary antibodies were incubated with 4◦C overnight
We employ CRISPR/Cas9 system into rat adipose-derived mesenchymal stem cells (Ad-MSCs) to interrupt kelch-like ECH-associated protein 1 (Keap1) gene so that Nrf2 can be released from ubiquitination and proteolytic degradation and works in the cellular nuclei
Summary
Like Alzheimer’s diseases, Parkinson’s diseases, stroke, epilepsy, and spinal cord injury, have become the greatest threats to the modern public health. Stem cell therapy is considered as an effective approach to repair tissue injury and treat these diseases [1,2,3,4,5]. Studies have shown that more than 99% cells died at 4–7 days in the post-grafting [6], because cells from injured tissue produce a large amount of reactive oxygen species (ROS) in hypoxic–ischemic environment, and the accumulation of ROS impairs stem cell survival after grafting [7, 8]. The inflammatory reactions induced by complex, variable, and hostile micro-environment in lesion areas become the culprits that torture the post-grafting stem cells to death. It is important to enhance the anti-oxidation and anti-inflammatory property after traumatic brain injury. This effort may ensure higher success rate and therapeutic effect of mesenchymal stem cell grafting
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