| Objective To extract exosomes from the supernatant culture of mouse bone marrow mesenchymal stem cells adherently cultured by whole bone marrow by ultracentrifugation to intervene in cerebral hemorrhage mice,by western-blot method,immunohistochemistry method,and animals Behavioral identification and other means to clarify that BMSCsderived exosomes regulate the mitochondrial function around the cerebral hemorrhage in mice and the effect on the improvement of nerve function.Methods Six to eight?week?old male ICR mice were randomly divided into 3 groups of 15 mice: 1)sham operation group 2)cerebral hemorrhage model group 3)exosome intervention group(2mg/kg)and marked with corresponding ear tags after modeling,Routine rearing,and the following operations: 1 through the stereotactic injection of autologous blood to establish a model of cerebral hemorrhage in mice.2 Extract and culture the primary BMSCs of young mice by whole bone marrow adherence method.3 Use adipogenic,chondrogenic,and osteogenic bone marrow mesenchymal stem cells to stain and identify them,and use flow cytometry to identify the phenotype of thirdgeneration cells.4 Extract the exosomes derived from bone marrow mesenchymal stem cells by using the improved differential ultracentrifugation method and use transmission electron microscopy,particle size detection,and Western blotting to identify.5 The brain edema of laboratory animals was tested by using the wet and dry specific gravity method.6 Morris water maze detects changes in animal behavior.7 Flow cytometry to detect changes in mitochondrial Ros.8 Detection of mitochondrial membrane potential of tissue cells.9 Determination of tissue ATP(adenosine triphosphate,ATP)level.10 Western-blot method to detect the effects of SPRR1 A and GAP-43 protein expression.11 Immunohistochemical method was used to detect the effects of SPRR1A(Small Proline Rich Protein 1A,SPRR1A)and GAP-43(Growth associated protein-43,GAP-43)protein expression.Results 1 The forelegs of the paralyzed side of the rat were recovered and flexed under the abdomen with tremor.When walking,there were signs of lateral rotation to the paralyzed side,accompanied by mental depression.After dissection of the brain of the model rat,comparing the left and right hemispheres,the right side is hyperemic,the sulci are disappeared,and the cerebral edema is severe.The shape of the third generation is round or shuttle-shaped,with strong refractive power and growing in a spiral shape.3 Alizarin red staining showed red with different depth after osteogenic induction,and blue precipitate appeared in the cytoplasm after chondrogenic induction.After induction of fat formation,the fat droplets were stained dark red with Oil Red O.Flow cytometry identification of bone marrow mesenchymal stem cells PE-CD90+,PE-CD54-,FITCCD29+,FITC-45-.4 After ultracentrifugation,the sediment under electron microscopy showed vesicles with a complete saucer structure;CD9 and CD63 were positively expressed,and Calnexin was negatively expressed.Nano2000 was used to determine the concentration of exosomes 40ug/ul.5 The dry and wet specific gravity method was used to measure cerebral edema and no significant change in the water content of the brain tissue in the sham operation group was found.In the model group,the water content of the brain tissue increased significantly within 1,3,and 7 days of modeling.The water content of brain tissue in the treatment group for 1,3,and 7 days was lower than that of the model group,and the difference was significant.6 The latent period of searching the safe island on the 7th,14 th and 21 st day after injury in the model group was significantly higher than that in the sham operation group,and the difference was significant.In the treatment group,the latent period of searching for the safe island on the 14 th and 21 st days after injury was significantly shorter than that of the sham operation group,and the difference was significant.7 Using the ROS value of the brain tissue of the sham-operated group as a control,the ROS amount of the brain tissue of the model group reached the peak peak right on the third day.The peak ROS of the brain tissue of the treatment group was left-biased compared with the peak value of the model group.The injury improved after treatment.8 Taking the peak of brain tissue membrane potential of mice in the sham-operated group as a control,the left-biased peak of the mitochondrial membrane potential of the brain tissue cells of the model group was lower than that of the sham-operated group,and the righthand peak of the mitochondrial membrane potential of the brain tissue of the treatment group was higher than the right For the model group,the difference is significant.9 The ATP of the model group showed that the injury could reduce the energy metabolism of the brain.The ATP content of the treatment group was lower than that of the sham operation group,but higher than that of the model group,and showed an upward trend.10 Westernblot test results showed that the expression of GAP-43 on the third day after exosome transplantation was higher in the model group than in the sham operation group;there was no significant difference in expression between the treatment group and the model group.On the 7th,14 th,and 21 st day after the exosome transplantation,GAP-43 model group and treatment group had higher expression compared with sham operation group;treatment group had higher expression of GAP-43 compared with model group..The expression of SPRR1 A was expressed on the third day after exosome transplantation.There was no statistical difference between the transplantation group and the model group.On the 7th,14 th,and 21 st day after exosome transplantation,the expression in the model group and the treatment group was higher than that in the sham operation group(P<0.05).Compared with the model group,the SPRR1 A expression was higher in the treatment group.Significantly increased.11 Immunohistochemical method detection: GAP-43 and SPRR1 A expression can be seen after exosome injection: GAP-43 and SPRR1 A are yellow or brown after staining.There is almost no positive expression in the brain tissue of the sham operation group and model group.The staining score was low;the positive expressions of GAP-43 and SPRR1 A in neurons were significantly increased in the treatment group,and the staining score was increased.The difference was statistically significant.Conclusions 1 The ICH model of ICR mice can be successfully established by autologous blood injection method and passed the behavioral test.Exosomes were successfully isolated from bone marrow mesenchymal stem cells by modified ultracentrifugation.Injecting exosomes through the nasal drip method has a certain protective effect on the mitochondrial function around the hemorrhage focus.The detection of GAP-43 and SPRR1 A protein proves that it has a certain role in neuroprotection after nerve injury.Figure17;Table8;Reference 93... |
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