| Objective: Spinal cord ischemia-reperfusion injury is a serious complication after surgery.The incidence of spinal cord ischemia-reperfusion injury is as high as 10-40%.Paraplegia or even death can be caused by severe cases,which greatly threatens the health and life of patients.With the continuous development of genetic engineering technology,stem cell transplantation for spinal cord ischemia-reperfusion injury has attracted wide attention.Exosomes,as an important medium for cell-to-cell communication,can bind to target cells through membrane receptors and participate in the regulation of biological information.Exosomes secreted by transplanted bone marrow mesenchymal stem cells(BMSCs)have been shown to play an important role in the repair of nervous system injury.In this study,rat bone marrow mesenchymal stem cells(BMSCs)were cultured in vitro.Bone marrow mesenchymal stem cells(BMSCs)stably transfected with miR-25 were obtained by gene transfection technology.Bone marrow mesenchymal stem cells(BMSCs)were injected intrathecally into the spinal cord of rats with ischemia-reperfusion injury.The protective effect of exosomes of bone marrow mesenchymal stem cells(BMSCs)overexpressing miR-25 on transient spinal cord ischemia Biotherapy for exosomes of systemic injury provides experimental ideas and therapeutic basis.Methods: 1.Rat bone marrow mesenchymal stem cells were resuscitated and subcultured.The morphology of cells was observed under optical microscope.Bone marrow mesenchymal stem cells were cultured and amplified by adherence method.Overexpression of miR-25 was constructed by lentivirus vector infection.Infection was observed by fluorescence microscopy,and the expression of miR-25 was determined by RT-PCR.2.Mass culture bone marrow mesenchymal stem cells,miR-25 expression-bone marrow mesenchymal stem cells and the control bone marrow mesenchymal stem cells by serum without exosomes and collecting cell culture supernatant,methods of collecting cell supernatant by ultracenfugation to extract exosomes derived from three style of cells,and the material with transmission electron microscopeto analyse the size and the morphology,western blot to detect the typical biomarker to identify extraction is exosomes.the expression of miR-25 was determined by RT-PCR.3.Rat spinal cord ischemia-reperfusion injury model was established by thoracotomy for 15 minutes with distal descending subclavian artery occlusion.Male SD rats were randomly divided into 5 groups.All rats except sham-operated animals were subjected to spinal cord ischemia for 15 minutes.(1)Sham group(n=11): Rats only underwent surgery without spinal cord ischemia.(2)Control group(n=11): One day before spinal cord ischemia,PBS(10uL)was injected intrathecally into each rat.(3)Exo group(n=12): 1 day before spinal cord ischemia,each rat was injected with bone marrow mesenchymal stem cell exosome(10 uL,2 mg/mL).(4)vector-Exo group(n=10): before spinal cord ischemia for 1 day,each rat was injected intrathecally with a control vector to infect the exosome of bone marrow mesenchymal stem cells(10uL,2 mg/mL).(5)miR-25-Exo group(n=11): 1 day before spinal cord ischemia,each rat was injected with pre-mir-25 virus into the sheath to infect the exosome of bone marrow mesenchymal stem cells(10uL,2 mg/mL).The motor Deficit Index(MDI)motor function scoring method was used to evaluate the motor function of hind limbs of 8 groups of animals 24 hours,48 hours and 7 days after reperfusion.The spinal cord tissues of 4-6 segments of the lumbar spine were taken for Nissl staining to count surviving neurons.Interleukin(IL)-1beta and tumor necrosis factor(TN)in spinal cord tissue were detected by ELISA.TNF-alpha content;Malondialdehyde(MDA)content and superoxide dismutase(SOD)activity were detected by kit;NOX 2 and NOX 4 protein expression were detected by Western blot;and the expression of miR-25 was detected by qRT-PCR.Results: 1.After resuscitation,culture and passage expansion,the morphology of rat bone marrow mesenchymal stem cells was spindle-shaped,which can be used as target cells for transfection.After screening with purinomycin,the level of miR-25 in bone marrow mesenchymal stem cells transfected with Pre-miR-25 lentivirus could be significantly increased(P < 001),but there was no significant positive expression in bone marrow mesenchymal stem cells transfected with empty vector.2.Under fluoroelectron microscopy,the exosomes were oval or circular membranous vesicles with a large number of contents in the vesicles,and the diameter was between 50 and 100 nm,which accorded with the structure characteristics and size of the exosomes.CD9,CD63 and CD81 were expressed in the three groups of cells.Transfection of pre-miR-25 lentivirus can significantly increase the level of miR-25 in the exosome of mesenchymal stem cells.3.1)There was no significant difference in body weight and rectal temperature between the five groups(P>0.05);compared with Sham group,the mean arterial pressure in the proximal and distal segments before blockade was significantly lower in the other groups(P < 0.05);2)motor function scores of hind limbs in rats: compared with the Control group,the MDI scores of Exo group,vector-Exo group and miR-25-Exo group were significantly lower,48 hours and 48 hours after perfusion.At 7 days,the MDI score of the miR-25-Exo group was significantly lower than that of the Exo group and the vector-Exo group(P=0.003,P=0.004).3)Histological examination of spinal cord: Nissl staining showed that the pathological changes of spinal cord tissue in the exo group,the vector-Exo group and the miR-25-Exo group were significantly improved compared with the Control group,and that in the miR-25-Exo group was the best,although the poliomyelitis in the Exo group and the vector-Exo group was the best.The number of surviving neurons was significantly higher than that of the control group(P< 0.001),but still the most surviving neurons were found in the miR-25-Exo group(P= 0.002).4)Contents of IL-1beta and TNF-alpha in spinal cord tissue: Compared with Sham group,IL-1beta and TNF-alpha increased significantly after reperfusion in Control group,Exo group,vector-Exo group and miR-25-Exo group(P<0.001),but there was no significant difference among the four groups(P>0.05).5)MDA content in spinal cord tissue: Compared with Sham group,MDA increased significantly after reperfusion in Control group,Exo group,vector-Exo group and miR-25-Exo group(P<0.001).The MDA content in Exo group and vector-Exo group was higher than that in control group,but there was no significant difference(P=0.233 and P=0.324).The MDA content in miR-25-Exo group was significantly lower than that in control group and Exo group(P<0.001 and P=0.022).6)SOD activity in spinal cord tissue :SOD activity in spinal cord tissue was significantly lower than that in Sham group(P<0.01).Only in miR-25-Exo group SOD activity was significantly increased compared with Control group(P = 0.03);7)The expression of miR-25 in spinal cord tissue: The expression of miR-25 in spinal cord tissue was slightly higher in Control group than in Sham group,but there was no significant difference(P>0.05).Compared with Control group,Exo group and vector-Exo group,the expression of miR-25 in spinal cord tissue was significantly higher in miR-25-Exo group(P < 0.01).NOX2 expression in tissues: Compared with Sham group,NOX2 expression in Control group,Exo group,vector-Exo group and miR-25-Exo group increased significantly after reperfusion(P=0.021,P=0.016,P=0.007,and P=0.002),but there was no significant difference in NOX2 expression in spinal ischemic tissue between the four groups(P > 0.05);9)NOX4 expression in spinal cord tissue: Compared with Sham group,NOX4 expression in spinal cord tissue was significantly increased in Control group,Exo group and vector-Exo group after reperfusion(P=0.001,P=0.003,P= 0.007).The expression of NOX4 in spinal cord tissue was significantly decreased in the miR-25-Exo group compared with the Control group and the Exo group(P< 0.002 and P = 0.012).Conclusions: Bone marrow mesenchymal stem cells(BMSCs)overexpressing miR-25 were successfully constructed by lentiviral vector infection.The exosomes could be successfully extracted by differential centrifugation.The expression of miR-25 in the exosomes of BMSCs could be significantly increased by lentiviral vector infection.Bone marrow mesenchymal stem cell exosome has neuroprotective effect on ischemic spinal cord,and the protective effect of bone marrow mesenchymal stem cell exosome overexpressing miR-25 on ischemic spinal cord is stronger.These evidences lay a foundation for further research on the proliferation of bone marrow mesenchymal stem cells transfected with miR-25 in vitro,stable expression and treatment of some related diseases. |
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