Font Size: a A A

The Mechanism Of Neuroprotective Efficcy Of MicroRNA-21 Overexpressing Bone Marrow Mesenchymal Stem Cells In Intracerebral Hemorrhage Rats

Posted on:2020-11-27Degree:DoctorType:Dissertation
Country:ChinaCandidate:H Y ZhangFull Text:PDF
GTID:1364330596995854Subject:Neurology
Abstract/Summary:PDF Full Text Request
Objective: Intracerebral hemorrhage(ICH)is a serious cerebrovascular disease with a high incidence of morbidity and mortality.Neurological dysfunction caused by ICH is mainly caused by degeneration,necrosis or apoptosis of the nerve cells around hematoma region after ICH.Studies have shown that hematoma formation after ICH can cause primary brain damage to the surrounding brain tissue,and red blood cells and their metabolites,necrotic cells and cytokines after ICH can trigger inflammatory reaction,affect blood coagulation and fibrinolytic system.These reactions can further aggravate the formation of cerebral edema around the hematoma,leading to more severe and persistent secondary damage.At present,patients with ICH have a poor prognosis and limited treatment.Therefore,it is important to selectively improve the treatment of ischemia,hypoxia,edema,and neuronal apoptosis around hematoma after hemorrhage.In recent years,bone marrow mesenchymal stem cells(BM-MSCs),as a stem cell with self-renewal ability and multiple differentiation potential,may play an important role in the treatment of ICH rats.Different from previous beliefs that BM-MSCs may play a neuroprotective role by differentiating into neural cells,more and more researchers found that BM-MSCs have limited ability to survive and differentiate in vivo,and more likely to release a variety of angiogenic and neuroprotective activities cytokines through paracrine secretion.In our previous studies,we found that microRNA-21(miR-21)was significantly decreased in peripheral blood and brain tissue of patients with ICH.Studies have shown that miR-21 plays an important role in the process of apoptosis,autophagy of spinal cord injury,tumors and other diseases,but its role in cerebral hemorrhage diseases is still unclear.This study intends to transfer rno-miR-21 to bone marrow mesenchymal stem cells,transplanted it into ICH animal models,and observe the effect of transfected stem cells on the recovery of neurological function,and its survival ability in ICH rats.The changes and the anti-apoptotic ability of peripheral nerve cells after hemorrhage may provide a new theoretical basis for the treatment of ICH by BM-MSCs.Research methods: In this study,Wistar rats weighing 250-300 g were made into ICH animal models by stereotactic brain collagenase VII injection.Bone marrow mesenchymal stem cells stably transfected with miR-21 by lentivirus were injected into the lesion by stereotaxic brain.The experiment was divided into sham operation group(Sham group),control group,MSC group,MSC-NC group and MSC-miR-21 group.The water content of brain tissue was measured,and the degree of edema of brain tissue was judged;Corner test was used.Neurological function scores were scored in rats with cerebral hemorrhage and fore limb placement test;The area size of ICH was measured by hematoxylin-eosin staining;BM-MSCs were stained by PKH26 live cell membrane dye,and was traced for the survival BM-MSC;Matrix metalloproteinase 2(MMP2),matrix metalloproteinase 9(MMP9),and tissue inhibitor of metalloproteinases 1(TIMP1),which the change in expression level determines the degree of brain tissue damage were detected by Western Blotting.Hemin was used to simulate the ICH environment in vitro.BM-MSCs were co-cultured with PC12 cells.Flow cytometry,TUNEL and Western blotting were used to detect apoptosis of cleaved Caspase-3.Exosomes were extracted by exosome extraction kit and Western blotting was used to detect exosomal marker proteins such as CD9,CD63 and CD81,and the exosome extraction was verified.The exosomes derived from BM-MSCs were labeled with PKH26,and the fusion of exosomes with neuron-like neurons was observed after coculture with PC12 in vitro.Online prediction of possible target genes for miR-21 by target gene prediction site,screening by real-time PCR,and direct binding of miR-21 to TRPM7 by dual luciferase reporter gene.Finally,by knocking down the expression of TRPM7,it was found that miR-21 may regulate the NF-κB pathway by regulating TRPM7 to protect the perihematomal neurons after cerebral hemorrhage.Results: 1.BM-MSCs overexpressed by miR-21 can significantly reduce the degree of cerebral edema in ICH rats(P<0.05).2.miR-21 overexpressing BM-MSCs can effectively improve the neurological function score of ICH rats(p<0.05).BM-MSCs improved the neurological function of ICH rats,but it was not statistically significant(p>0.05).BM-MSCs overexpressing miR-21 were more effective than BM-MSCs-NC(p<0.05).3.miR-21 overexpressing BM-MSCs can significantly reduce the hematoma area and brain tissue damage in ICH rats.4.miR-21 can effectively increase the survival rate of BM-MSCs in the brain of ICH rats.(P<0.05)5.miR-21 overexpressed BM-MSCs can reduce the apoptosis of perihematomal neurons after ICH,and the effect on astrocytes is not obvious.6.miR-21 can improve the anti-apoptotic ability of BM-MSCs under Hemin treatment conditions,but the proliferation ability has no obvious change.7.MiR-21-overexpressing BM-MSCs-derived exosomes can be membrane-fused with PC12 cells in vitro,and the anti-apoptotic ability of co-cultured neurons is significantly decreased after inhibiting exocrine secretion.The degree of recovery of neurological function in rats was weakened.BM-MSCs overexpressing miR-21 can decrease the expression of TRPM7 in PC12 cells and inhibit the apoptosis of PC12 cells in vitro by inhibiting NF-κB pathway.Conclusion: 1.miR-21 can effectively improve the neurological function of BM-MSCs in ICH rats.2.miR-21 can significantly improve the survival rate of BM-MSCs in the brain of ICH rats and the anti-apoptotic ability of hemin in the treatment of hemin.3.BM-MSCs can transfer miR-21 to neurons through exosomes,and directly inhibit the expression of TRPM7 by directly binding to the 3’-UTR region of TRPM7 mRNA in neurons,further inhibiting the activation of NF-κB pathway.Thereby achieving neuroprotection.MiR-21 overexpressing BM-MSCs up-regulate miR-21 in neurons by up-regulating miR-21-rich exosomes and significantly enhance neuronal anti-apoptosis via miR-21/TRPM7/NF-κB pathway The ability of miR-21 to enhance the neurological function of BM-MSCs in ICH rats may provide a new strategy for the treatment of ICH.
Keywords/Search Tags:Intracerebral hemorrhage(ICH), Bone Marrow Mesenchymal Stem Cells(BM-MSCs), microRNA-21, Exosomes, TRPM7
PDF Full Text Request
Related items