| Objective:To investigate the effect of Vimentin on the microtubules and neurite outgrowth.Methods:First,The model of spinal cord injury in rats was established by the device of Louisville damage system.Immunohistochemisty and Q-PCR were performed to observe th expression of Vimentin in the injured spinal cord.Then the eukaryotic expression vector Vimentin-p EGFP-C2 was constructed by the method of homologous recombination and the si RNA-Vimentin fragment was synthesized,the plasmid and interference fragment were transfected into HEK 293 T cells,and the proteins of the transfected cells were extracted for Western Blot experiment to validate its validity.Followed by overexpression and silencing of Vimentin,in COS7 cells,the changes of fluorescence intensity of ace-tubulin and Tyr-tubulin were observed by immunocytochemistry,and finally the changes of neurite length and number were observed by overexpression and silencing Vimentin,in hippocampal neurons.Results:1.The spinal cord tissue was taken for immunofluorescence experiment.The results showed that compared with the control group,the fluorescence intensity of Vimentin in the experimental group was stronger than that in the control group,and the increase correlated with the degree of spinal cord injury.Statistical analysis of QPCR results showed that the m RNA expression of Vimentin in the mild injury group was higher than that in the control group(P<0.05),the m RNA expression of Vimentin in the moderate injury group was higher than that in the control group(P < 0.05),and the m RNA expression of Vimentin in the severe injury group was higher than that in the control group(P< 0.001).2.The eukaryotic expression vector Vimentin-p EGFP-C2 was successfully constructed and verified,and the designed three interference fragments all had interference effect.3.Through overexpression and silencing of Vimentin,in COS7 cells,it was found that in the overexpression experiment,the fluorescence intensity of ace-tubulin/tyr-tubulin was significantly higher than that of the control group(P< 0.01).In the silence experiment,the fluorescence intensity of ace-tubulin/tyr-tubulin was significantly lower than that of the control group(P< 0.01).4.through overexpression and silencing of Vimentin,in hippocampal neurons,it was found that in the overexpression experiment,compared with the control group GFP,the total number of GFP-Vimentin,the number of primary branches,the number of secondary branches(all P<0.01),the length of total processes(P<0.05),the length of primary branches(P<0.01)and the length of secondary branches(P<0.05)increased in the experimental group.(figure 11),The second group: compared with GFP+NC group,the total number of GFP+si RNA-2 decreased(P<0.05),the number of primary branches decreased(P<0.05),the number of secondary branches unchanged,the total process length decreased(P<0.05),the length of primary branches decreased(P<0.01)and the length of secondary branches unchanged in the experimental group.Conclusion:1.Vimentin can increase the stability of microtubules.2.Vimentin can promote the neurite outgrowth in rat hippocampal neurons. |
