| Objective:To investigate the correlation between the mTOR signaling pathway and the murine models of malformations of cortical development(MCD)induced by X-ray irradiation in pregnant mice,and to explore the protective effect and possible mechanism of rapamycin on the brain development.Methods: 1.The pregnancy SD rats were randomly divided into normal group,model group,DMSO group and rapamycin group,and was given the corresponding pretreatment consecutively on the E16 and after.Among them,model group,DMSO group and rapamycin group were treated with X-ray irradiation on E17,and the first generation offspring(F1 generation)were used as experimental subjects to establish the model of malformation of cortical development.2.Then the rats of F1 generation in each group were randomly selected 18 experimental mice in birth day,the first day,the second day,the third day and the tenth day(P0,P1,P2,P3,P10),then decapitated brain tissue.3.Six of them were immediately fixed in 4% paraformaldehyde,then measured the anteroposterior diameter,upper and lower diameter,left and right diameter size of brain tissue to analysis the volume differences,Then,the brain tissue by a certain percentage of pathological sections,and finally the pathological changes of brain were analyzed by HE staining and Nissl staining.4.The remaining 12 brain tissues were immediately frozen in-80? refrigerator,and to detected the expression of S6 mRNA and S6 protein in the brain tissue of rats by qPCR and by Western blotting respectively in P0,P1,P2,P3 and P10 all 5 days.Results: 1.After roughly observed the experimental brain tissue and measured,In the normal group,the anterior and posterior diameters of brain tissue at various observation time points,and the up-and-down diameter of brain tissue except P0 at the time point were higher than that young rats born after X-ray irradiation(model group,solvent group and rapamycin group),the difference was statistically significant(p<0.05),there was no significant difference in the brain tissue of neonatal rats after X-ray irradiation(p>0.05).But overall: we can found the model group was smaller than the normal group,the DMSO group is similar to the model group,but the rapamycin group size was close to the normal group.2.Optical microscope observation suggested that under the cortical and subcortical conditions,the structure of hippocampus was clear and the dendrites were clear,the neurons were arranged neatly.There were no abnormal size and shape of neurons and corpus callosum in the normal group.The cortical structures of the model were disorder,corpus callosum loss,cortex,hippocampus can be abnormal nodules,neurons arranged in an uneven,polar disorders,etc.There was no significant change in the pathological changes between the DMSO group and the model group,the rapamycin group were observed the above change,but were more normal than the model group.3.qPCR showed that the S6 mRNA detection in the model group was higher than that in the normal group in each observation time point(p <0.05),there was no significant difference in the expression of S6 mRNA between the DMSO group and the model group(p>0.05),while the expression of S6 mRNA in rapamycin group was lower than that in model group,still slightly higher than that in normal group.4.Western blotting showed that the expression level of S6 protein in the model group was significantly higher than that in the normal group starting from P0(p <0.05),there was no significant difference in the expression level of S6 protein between DMSO group and model group(p>0.05),while the expression of S6 protein in rapamycin group was lower than that in model group,and higher than the normal group,the difference was statistically significant(p <0.05).Conclusion: 1.Confirmed that the pilot study of this experiment obtained 195 cGy dose of X-ray irradiation pregnant 17 days after the birth of the child can be successfully established MCD model results,this method is simple and high success rate.2.In this experimental model,it was confirmed that there is a close correlation between the mTOR signaling pathway and MCD mice induced after X-ray irradiation.3.Rapamycin has a certain degree of protective effect on the development of the brain tissue of the F1 generation of pregnant rats after X-ray irradiation,its mechanism may be related to rapamycin can inhibit the abnormal activation of mTOR signaling pathway,so that MCD brain tissue development is improved. |
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