| Objective To investigate whether Mex3c gene-deficient mice involved in regulating the energy balance of the body by inducing the expression of c-fos in the hypothalamus and observe whether there are defects in the development of neural tubes in the embryonic mice of Mex3c offspring.In order to provide a the oretical basis for later research on the role of Mex3c in neural tube development.Methods?1?Using CRISPR/Cas9 gene editing technology,designed of Mex3c gene sgRNA,coinjected mouse mex3c gene gRNA and Cas9 mRNA into fertilized eggs to generate the target gene knockout offspring,three weeks after the birth of offspring mice,we extract DNA to identify mouse genotypes.The obtained F0 generation heterozygous mice continued to breed with normal group mice to obtain F1 and 2 generation mice,then isolate heterozygous mice for experiments.?2?8-week-old normal group,(Each group was randomly divided into normal control group?normal saline 1?L·g-11 weight,N=10?,normal group leptin group?leptin 5?g·g-11 weight,N=10?,normal group ghrelin group?ghrelin 1?g·g-11 weight,N=10?,Mex3c-/+control group,Mex3c-/+leptin group,Mex3c-/+ghrelin group,Mex3c-/+mice were injected and the same amount as normal group,each group of mice deal with intraperitoneal injection.H&E and Nissl staining were used to observe the morphology of hypothalamic nerve cells.The expression of c-fos in the hypothalamic nucleus of each group of mice was analyzed by immunohistochemical techniques.?3?Normal and Mex3c-/+female mice were fed with normal diets?10 mice per group?,10 female mice on a high-sugar and high-fat diets.Each group started at 8 weeks of age and continuous feeding for 12 weeks then copulate with normal male mice to obtain E14.5-day embryos from each group.H&E staining was used to observe neural tube morphology,LFB staining was used to observe nerve myelin sheath,TEM was used to observe neuronal ultrastructure and immunohistochemical techniques were used to analyze nestin expression.Results?1?Successfully construct Mex3 gene-deficient mice whose genes can be stably inherited.?2?H&E and Nissl body staining results showed no significant differences in pathological staining between normal mice and Mex3c-/+mice.Immunohistochemical results showed that the expression of c-fos protein in hypothalamus nerve nucleus of normal control mice was low but the expression of c-fos was significantly increased 2h after inection of leptin and ghrelin?*P<0.05?;Mex3c-/+control mice had higher expression of c-fos than normal control mice?*P<0.05?and there was no difference compared with normal leptin group mice?P>0.05?;The intervention of ghrelin in Mex3c-/+mice,the expression of c-fos was significantly down regulated which was basically the same as that in the normal control group.There was no significantly different between normal leptin,ghrelin group,Mex3c-/+control and leptin group in c-fos expression.?3?In the neural tube study,we found no difference in the embryos of each group by H&E staining.Myelin staining can be seen in the normal group which is obviously blue and darker.Considering the result of higher color rendering conditions due to the low degree of myelin development.From TEM observation,compared with the normal group,Mex3c-/+and HD groups showed a large number of vacuoles,the mitochondrial cristae were broken and the structure was significantly abnormal.The presynapticmembrane and postsynaptic membrane is clear but the synapticspace was disappeared,the synapses have a certain degree of fusion and the typical structure is difficult to find.Nestin results show lower expression in the normal group than HD and Mex3c.Conclusion Mex3c is involved in the process of energy regulation in vivo by elevated the expression of c-fos in the hypothalamus.In addition,imbalanced energy balance causes female mouse embryonic neural tubes to develop later than normal mice in normal development. |
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