مقالات پذیرفته شده در سومین کنگره بین المللی زیست پزشکی
Are modified-astrocytes by deadCas9 a new therapy of chronic ischemic stroke in the Rat?
Are modified-astrocytes by deadCas9 a new therapy of chronic ischemic stroke in the Rat?
Melika Lotfi,1,*
1. School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
Introduction: The number of stroke-based decease is enhancing, and stroke stays as one of the leading causes of decease and incapacitation all around the world. The two chief sorts of stroke have been identified, ischemic and hemorrhagic. However, for neither of them, there are some definitive FDA-approved (food and drug administer) treatments without any side effects. So the research for new therapies is significant. The objective of this hypothesis is to improve the complications of chronic ischemic stroke in the induced Sprague-Dawley (SD) rat model by Intraluminal suture MCAo (middle cerebral artery occlusion) utilizing combining cell therapy and gene therapy. Both methods are the latest treatments for improving the complications of the stroke, but there are some problems with each one that can be solved by this hypothesis. Many pieces of evidence suggest that astrocytes are as an appropriate option for cell therapy of stroke due to their multiple functions in the CNS. Based on the Stroke Treatment Academic Industry Roundtable stroke is one of the complex illnesses. Therefore, we propose to modify ex vivo astrocytes from the rat models by transducing a gene profile including IL-38 as an anti-inflammatory agent, BRAG-1 as an anti-apoptotic factor with the best genome editing tool up to now, CRISPER deadCas9.
Moreover, astrocytes have a significant role in the regulation of plasminogen activation in CNS by providing a surface for tissue plasminogen activator, which stimulates pro-BDNF (brain-derived neurotrophic factor) and fibrinogen devastation. It prepares the ischemic area for the natural migration of NSC (neural stem cell) which can enhance motor function improvement without any side effect; also they have a crucial role in angiogenesis by secreting VEGF which helps with BRAG-1 in preventing apoptosis and hypoxic death of themselves and migrant NSC. By this way, it is expected to have the multiple therapy for chronic ischemic stroke
Methods: Extracting astrocytes from the brain of SD rat models.
Inducing chronic ischemic stroke in the SD rat models by Intraluminal suture MCAo (middle cerebral artery occlusion)
•Constructing the gene profile including IL-38, BRAG-1, VP64-dSpCas9 gene, 2complementary gRNA sequences for IL-38, BRAG-1 employing the deadCas9-VP64-GFP plasmid which is about 14,547bp.
•Transfecting the gene profile plasmid into the ex vivo astrocytes by Electroporation as it is fast, easy, with high efficiency, and can be used for numerous cell types.
•Identifying the gene profile in the astrocytes by PCR.
•Culturing the modified astrocytes.
The most popular manner is Flow Cytometry sorting, Percoll Gradient, and Mixed Primary glial culture in rats.
•intracranial injection of the cultured genetically modified astrocytes into the ischemic region and its surrounding area in the brain of SD rat models.
•RT-PCR is accomplished to gauge the level of IL-38, and BRAG1 and ELISA are performed to measure the level of IL-38, IL-22, IL-8, IL-17.
•There are two groups of rat models; the first one contains three control animals that are not injected, the rest of them are fall into three categories of 3 animals that each group is injected by different modified astrocytes based on altered states of transducing gene profile and VP64-dSpCas9 with complementary sgRNAs.
•Investigating the results of the functions of different modified astrocytes based on the different expression of transducing gene profile and VP64-dSpCas9 with complementary sgRNAs in ameliorating the chronic ischemic stroke deficits in the rat model by MRI assessing.
•The Bederson scale for testing motor function, which is a global neurological assessment that was developed to measure neurological defects following stroke
Results: It’s expected that this proposal can alleviate the deficits of chronic ischemic stroke in the SD rat model of chronic ischemic stroke as it is a multiplex therapy which can inhibit inflammatory functions by expression of IL-38, prepare the area for migration of NSC without tumor genesis, and preventing neural cell, migrant NSCs, injected modified astrocytes death by expression of BRAG-1 gene by means of the most accurate genome editing tool CRISPER I ( inhibitor) attached to VP64 with the least side effects as all the system can be switched-off by anti-CRISPER proteins if rare adverse effects occur.
Conclusion: As a conclusion, combined cytokine gene therapy and cell therapy based on ex vivo modifying astrocytes by transfecting a gene profile including IL-38, BRAG-1 with the best genome editing tool up to now, VP64-dSpCas9 one form of CRISPER system, is expected to demonstrate safety and effectiveness in improving post-stroke deficits in chronic ischemic stroke.
Another study must be done on the combination of the function of the crisper system with another science like Epi genetic in ameliorating the deficits of stroke as well as evaluating the effects of using multiplex CRISPER in stroke therapy.