| Intracerebral hemorrhage(ICH),with high morbidity,mortality and disability rate,has become an urgent problem that needs to be solved urgently.Finding a certain drug to repair nerve cell skeleton and axon after ICH can provide a new target for clinical treatment of intracerebral hemorrhage.Bone marrow-derived mesenchymal stem cells(BMSCs)is a kind of stem cells.In recent years,attention has been paid to the role of BMSCs after ICH in the damage and repair of axon and cytoskeleton after ICH.Objective: After intracerebral hemorrhage(ICH)rats were given BMSCs and 3-MA.to observe the changes of the expression of neurofilament200NF-200,microtubule-associated protein-2 MAP-2and autophagy protein LC3,and to explore the effects of BMSCs and autophagy on neuronal cytoskeleton and axon after intracerebral hemorrhage.Methods:1 Ten 3-week-old Sprague-Dawley(SD)male rats were isolated by BMSCs and cultured.The three week-old female rats were cultured by adherent method in vitro.Cell growth was observed and cultured.Flow cytometry was used to detect BMSCs surface markers.2 The 4 group method 120 SD female rats were randomly divided into two groups: group Sham(n=30),ICH group(n=30)and ICH+BMSCs group(n=30)and group ICH+BMSCs+3-MA(n=30).ICH,ICH+BMSCs,ICH+BMSCs+3-MA group using stereotactic instrument model,enter 100 microliters of autologous blood,BMSCs and 3-MA.Sham group did not.Detected by bleeding after 1,3,5,7d were detected,the area around the hematoma with HE staining;the water content The protein expression of NF200mMAP-2mLC3 was detected by specific gravity immunohistochemicalmethod and LC3 localization 4 Western-blot.The water labyrinth test was performed on the other 40 rats 7~10 days after intracerebral hemorrhage.The statistical results were analyzed by software SPSS21.0.Q test was used to compare the statistical results.The difference between the two groups was measured by ANOVA.The statistical significance was P < 0.05.Results:1.The results of BMSCs culture and detection: about 11~15 days after passage,the cells were transferred to the third passage,and the cells gradually fused.The results of flow cytometry showed that the positive rate of BMSCs surface markers was 75.63% and 75.63% respectively.The positive rate of CD106 and CD29 was 76.477.The positive rate of CD45 was 5.39 and the positive rate of CD54 was 4.61%.The positive rate of CD45 and CD54 were negative.2.The results of HE staining showed that the brain tissue of the rats in the group of: Sham was complete,and the number of brain tissue was more than that in the group of nuclear integrity.ICH group was not closely arranged after intracerebral hemorrhage,prominent edema,capillary dilatation,nuclear defect.ICH BMSCs group compared with the ICH group,the vascular gap was enlarged and edema was reduced.Compared with ICH BMSCs group,the number of cells in ICH BMSCs 3-MA group was lower than that in ICH BMSCs group.3.The results of water maze test: compared with the Sham group,it took longer for the rats in the ICH group to find the target quadrant(P < 0.05).Compared with the ICH group,the time required to find the target platform in the ICH BMSCs group 7~10 days after bleeding was shorter than that in the ICH BMSCs group,P < 0.05,compared with that in the ICH BMSCs group.The time of finding platform in ICH BMSCs 3-MA group was prolonged 7 to10 days after bleeding(P < 0.05).4.There was no difference in the water content of brain tissue in the group of brain water content of 1,3,5 and 7 days,and the water content of brain tissue in the ICH group increased from the first day to the third day,reached the highest value on the third day,and then began to decrease,and the water content of the brain tissue in the ICH group was lower.The water content of ICH BMSCs group was higher than that of Sham group.The water content of ICH BMSCs group was lower than that of ICH group at 1-7 days.Compared with ICH BMSCs group,the water content of ICH BMSCs 3-MA group was similar to that of ICH BMSCs group,and the water content of ICH BMSCs 3-MA group was higher than that of ICH BMSCs group at 1~7 days,and the water content of ICH BMSCs 3-MA group was higher than that of ICH group at 1~7 days.5.The results of MAP-2 Western blotting showed that the expression of MAP-2 was similar among the sham operation groups,and the expression of MAP-2 protein in the strongly expressed group began to decrease at 1 day after bleeding(P < 0.05),then decreased to the lowest on the third day,and then began to rise again.However,the expression of MAP-2 in the BMSCs group was lower than that in the Sham group(P < 0.05).ICH BMSCs had the same trend as that in the ICH group.However,each time point was higher than that in the ICH group,and the difference between 1~7 days after hemorrhage was more significant than that in the BMSCs group(P < 0.05).The expression of SCs 3-MA was the same as that of the former at all time points,but its content was lower at each time point(P < 0.05).6.The results of NF200 Western blotting showed that the expression of NF200 was similar in sham-operation group,and the expression of NF200 in both groups began to decrease at 1 day after bleeding(P < 0.05),decreased to the lowest at 3 days,and then began to rise again.However,the expression of NF200 in the BMSCs group was lower than that in the Sham group(P < 0.05).ICH BMSCs had the same trend as that in the ICH group.However,each time point was higher than that in the ICH group,and the difference was more significant at 1~7 days after hemorrhage than that in the BMSCs group(P <0.05).The expression of H BMSCs 3-MA was the same as that of the former at all time points,but the content of H BMSCs 3-MA was lower than that of the former at each time point(P < 0.05).7.The results of LC3 Western blotting analysis showed that there was no significant difference between LC3 ? / LC3 ? group and Sham group in LC3 ? / LC3 ? group.Compared with Sham group,the positive degree of ICH group increased at every time.The rise began on the first day(p < 0.05),and the peak occurred on the third day(P < 0.05).The whole process positive rate was higher in ICH BMSCs group than in Sham group.The whole process positive degree of ICH BMSCs group was higher than that in ICH group.Compared with ICH group,the change trend of ICH BMSCs 3-MA group and ICH BMSCs group was the same,and the expression of ICH BMSCs 3-MA group was slightly lower than that of ICH group at all time points(P < 0.05).8.The results of LC3 immunohistochemistry: the positive cells in the cortex around hematoma were detected in the LC3 Sam group.Compared with the Sham group,the number of LC3 cells in the ICH group was higher than that in the Sham group,and the peak value was the same as in the ICH group and the ICH group on the 3rd day.The positive rate of ICH BMSCs 3-MA group was weaker than that of ICH BMSCs group(P < 0.05),and the trend of change was similar(P < 0.05).Conclusion: After intracerebral hemorrhage,stereotactic injection of BMSCs can improve neural function and spatial exploration ability by activating autophagy activity,and can recover injured neurons,axons and skeleton proteins.And autophagy played a positive role in promoting it. |
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