| The traditional view in the field of neuroscience is that mature neurons in the adult mammalian CNS are terminally differentiated cells and no longer have the ability to divide and proliferate.In some studies,there are signs that neurons in the central nervous system can divide.However,these studies lack key evidence.The question of whether mature neurons can return to the cell cycle for mitosis has always been the focus of neuroscience.After more than ten years of exploration in our laboratory,we found that a modified neurotrophin/growth factor cocktail(patent number ZL200710063180.8)with T3(3,5,3?-triiodothyronine)can induce primary cultured rat cortical neurons divide,and through a large number of experiments confirmed that T3 can affect E2F1,Rb and other cytokines by down-regulating the expression of Necdin,and then participate in cell cycle regulation,and ultimately induce neuronal division.Later,through continuous improvement of this kind of cocktail,it was also successful in animal experiments.The cocktail was injected into the rat cortex by means of microdialysis and stereotaxic injection.After immunofluorescence labeling,a large number of cortical intrinsic neurons were found to be divided.Based on the previous work,this study will use proteomics techniques and animal behavioral experiments to continue to explore the effects of the cocktail of T3 on animals.In the first part of the experiment,open field experiments and shuttle box experiments were used to evaluate the behavioral changes of the animals after cocktail intervention.On the basis of previous experiments such as immunofluorescent labeling,it was found that there are a large number of neurons in the vicinity of the cocktail injection area,and the influence of this histological change on the whole animal needs the more intuitive index of behavioral experiments to evaluate The open field experiment and shuttle box experiment are widely used in the field of neuroscience and can better reflect the effect of cocktail intervention.We used 20-month-old aged rats to perform experiments.We used microstereoscopic techniques to inject 1/5concentration of cocktails into 4 spots on the left and right sides of the cortex of the aged rat cortex,followed by continuous ethological observations.After 150 days of consecutive observations,behavioral data were obtained,and behavioral indicators before the cocktail injection were compared with another normal saline injection group.The “total travel distance” and “center area travel distance” in the open field experiment were selected,and the “non-response times” in the shuttle box experiment was displayed as the evaluation index,and the mean±standard deviationwas used to show the variance analysis.Methods such as the differential test of the experimental groups.Through analysis of variance,we found that the “total moving distance” in the open field experiment of experimental animals began to increase 35 days after cocktail injection,and there was a significant difference between the injection and the injection before injection.The 35 th day after the injection and saline injection.There was a significant difference afterwards,indicating that the intervention of cocktails has increased the exploration and athletic ability of experimental animals in the new environment.After the cocktail injection,the “center movement distance”was also increased compared with that before the injection and the saline control group,but it failed to be reflected in the statistical difference.Shuttle box experiments need to train animals before cocktail injection.Animals should escape in time after acousto-optic stimulation in order to avoid receiving small doses of electrical stimulation.“No response times” means that animals do not escape after a preset stimulus is sent out.The number of reactions can reflect the animal's ability to learn and remember to a certain extent.After the cocktail injection,before the cocktail injection and the saline injection group,the “non-response frequency” of the animals after the cocktail injection was decreased,suggesting that the animals after the intervention had a certain degree of learning and memory capacity.It is worth pointing out that the differences between the individual bodies of older rats are large,and the difference in body weight between older animals is large,and some animals suffer from diseases due to old age.The standard deviation of measured data is relatively large.Experiments should be conducted.Increase the sample size to more accurately describe the behavioral changes after cocktail intervention.In the second part of the experiment,we used proteomics to analyze the effects of T3 and cocktail intervention on rats.Adult SD rats were selected for the experiment.Normal saline,T3 and cocktail were injected into the rat cortex M1 by microstereotactic technique.The cocktail was injected at 1 point of the left and right brain of the cortex M1.T3 was injected into the left cortex M1.In the area,saline was injected into the M1 area of ??the right cortex,and the coordinates of the injection point were symmetrical.The tissues around the injection area were removed at 6hours,1 day,and 2 days after injection,and the tissues were divided into 12 groups according to different injections,different injection positions,and different time after injection.Samples for protein proteomics were co-quantified to 4702 proteins by sRP technology and high-throughput mass spectrometry platforms.And iBAQ(intensity based absolute quantification)based on the peak area of ??the non-standard quantitative characterization of all protein expression levels,through log2 logarithmictransformation after the processing of the missing value and the application of the Shapiro-Wilk test on the normality of the data test.After normalization using the Width adjustment method,screening for different regions of the protein(RE)was performed.The screening conditions were statistically different for each group and the other 11 groups(p-value< 0.05),and the average expression level should be more than5 times.There were 505 differential proteins between T3 and cocktail and saline control at each time point,of which 288 were up-regulated in T3 or cocktail group and217 were down-regulated.Pearson correlation analysis was used to evaluate the correlation between groups.The Pearson correlation coefficient fluctuates between0.461 and 0.890.The correlation between the left and right sides of the cocktail treatment group at each time point is strong.Each time point T3 and cocktail The treatment was associated with a lower level of saline treatment,suggesting that there was no significant difference between the right and left sides in the cocktail injection of this experiment,but there was a better difference between the two treatment groups compared with the saline control group.The results of principal component analysis and cluster analysis also confirm the above conclusions.At the same time,it is found that the grouping factor of time plays an important role in the whole analysis.The following GO function annotation results showed that there were differences in the biological processes of each group.According to T3 and cocktail saline group,the differential proteins were significantly enriched to 129 items of GO biology,of which 70 were up-regulated and down-regulated.There are 59,involved in a number of important biological processes including cell proliferation and differentiation,nervous system development,embryo development,Hippo signaling pathway,Wnt signaling pathway.The biological processes involved in embryonic development and nervous system development can provide insights into the mechanisms that induce the division of mature neurons,and significantly enriched Wnt signaling pathways and Hippo signaling pathways are involved in the occurrence,development,and damage of the nervous system.Rehabilitation and other important processes.Preliminary analysis of proteomics provides insights into the mechanism of cocktail-induced cleavage of mature neurons using T3 as the main component. |
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