| Parkinson’s disease (PD) is a common age-related degenerative disease of the central nervous system. So far, the pathogenesis of PD is still not clear. Among them, the dysfunction of relationship of PD and apoptosis and the ubiquitin-proteasome system (ubiquitin proteasome system, UPS) dysfunction becomes a research hotspot to the pathogenesis of PD in recent years. Ndfipl protein was named by specific binding with Nedd4protein, and the present study has shown that Ndfip1protein has a outstanding effect in the protection of nerve cells. This topic used MPP+(1-methyl-4-phenyl-5-4hydrogen pyridine) which is the current relatively widely used for PD model was used in this research to induce the PD model of cultured human neuroblastoma SH-SY5Y cells and the embryo midbrain dopaminergic neurons of mice. Lentiviral vectors were used to transfer steadily Ndfip1. The role of Ndfip1in neuro-protection was studied these cell model induced by MPP+and the function of Ndfip1in regulating ubiquitination was also measured.The first part, the amplification, extraction and identification of the Ndfipl lentiviral vector and some related plasmids.Firstly, the presence of the enzyme of the plasmid which containing Ndfipl:pFU5xUAS mm Ndfipl C-Flag SV40Puro carrier (no.8599) was done to identy the existence of the Ndfipl DNA. And then the plasmids related with the package of virus and other plasmids were transformed respectively into the competent of escherichia coli bacteria, after that we took the monoclonal, shaked the bacteria, got the plasmid, did electrophoresis appraisal of agarose gel for the purpose of getting the plasmids with relatively high quality and concentration.The second part, the virus particles was packaged and four cell models were established.293T cells were used to package virus particles which were used to infect SH-SY5Y cells, and then antibiotics of hygromycin and puromycin were used to screen SH-SY5Y cells. Western Blot test was used to confirm that we had got many positive clones which could express Ndfip1stably. Then4HT was used to induce the overexpression of Ndfipl.The pGIPZ shRNA for Ndfip1was used in the transient transfection of the stable cell lines. In the case of adding4HT, the expression changes of Ndfip1protein were tested, and finally the effect of shRNA inhibiting(knock down) the action of the Ndfip1expression was confirmed. The acquiration of the cell model of dominant negative expression (4HT induce, the dominant negative carrier pFU mm Ndfip1Y42A67A EGFP SV40puro W (Ubiquitin Promoter) also used transient transfection. Thus four different cell models were built successfully:endogenous expression of Ndfip1(WT), over-expression of Ndfip1(4HT induced), dominant expression of Ndfip1(dominant negative carrier) and inhibition of negative expression of Ndfip1(pGIPZ shRNA for transient transfection of Ndfip1).The third part, Ndfip1plays a protective role in the process of cell damage and apoptosis of SH-SY5Y cells induced by MPP+.First of all, on the basis of the four cell models above, MPP+(1-methyl-4-phenyl-5-4hydrogen pyridine) was used to induce cells to be the PD model, then we conducted the research of the protection of Ndfipl with cell injury and apoptosis. Removing growth factor was used to induce cell apoptosis, the results from MTT test and flow cytometry showed that the expression of Ndfip1could protect SH-SY5Y cells and reduce apoptosis. While dominant negative and weak expression showed that he ability to protect cells against apoptosis decreased obviously which showed that restraination and reduction expression of Ndfip1could not protect cell.Later, the ubiquitin antibody was used to dectect the procee of ubiquitination, the results showed that the protection of Ndfip1follows with much ubiquitination of a variety of proteins which suggests the molecular mechanisms of the protection of Ndfip1involved varieties of ubiquitination of proteins. Precipitation and Western Blot were used to detect Nedd4-1and Itch which are the members of Hect family proteins and the results showed that the protection of Ndfip1may be caused by combining with Nedd4-1,because Nedd4-1is one E3ubiquitin ligase. SO Ndfip1could adjust apoptosis, etc. may combine with Nedd4and collect a variety of proteins which would be ubiquitined, which is consistent with the result of ubiquitination increasing,and the results also suggested that Ndfip1may bridged a variety of ubiquitin proteins and played an important role in the process of many kinds of cells. In the experiment, another member (4HT) of HECT family was also tested, with the time going on of adding4HT, the expression of Ndfipl gradually increased until to maintain a high expression level, meanwhile the expression of Itch gradually declined which suggested that the protection of Ndfip1in nerve cells may be associated with the expression decreased of Itch. Our team also studied the relationship between Ndfip1and p-Erk and the results showed that the effect of Ndfip1follows with lower levels p-Erk, and those showed that the protection of Ndfipl in nerve cells related to the signal transduction pathway of Erk.The fourth part, Ndfipl plays a protective role in the process of PD model which was induced by MPP+on primary cell of mouse embryo midbrain neurons.When the primitive cells of embryo midbrain neurons in mice were prepared, with MPP+induced its apoptosis, then one plasmid of Ndfip1was transfected into it by which we could study if there is a protective effect on neuronal apoptosis. The results show that the MPP+can induce its apoptosis, and compared with control group, the apoptosis of the number of mouse embryos nerve cells decreased significantly when Ndfipl protein over-expressed in the experimental group. Those results show that the overexpression of Ndfipl may inhibite the apoptosis of primitive cells of mouse embryonic midbrain neurons and reduces the number of cell apoptosis and has certain protective effect on the nerve cells which lay the experimental foundation for further study of the protection of Ndfipl in PD.Above all, Ndfipl can significantly inhibit the apoptosis of SH-SY5Y cell which induced by methods like the removing of growth factor, so Ndfipl protect nerve cells. And this effect with the ubiquitination of a variety of proteins and the combination with the proteins such as Nedd4etc of the Hect family, meanwhile following with the declining of the expression of Itch and the decreasing of the level of Erk with time extending. These results suggest that Ndfipl can protect nerve cells obviously, and the process is closely related to the ubiquitination of some proteins and its targets were related to Itch and the Erk signaling pathway. The PD model of primitive cells in mice embryonic midbrain neurons showed that overexpression of Ndfipl has a inhibition on the apoptosis of mice embryonic midbrain neuron cell again and it can effectively reduce the number of cell apoptosis and has certain protective effect on the nerve cells. The two different cell model of PD induced by MPP+, for the first time to discuss the molecular mechanism of Ndfpl of the protective effect on nerve cell and expound the biological significance of Ndfipl. Our results provide a basis for further research targets of Ndfipl and the related signal transduction pathways and the carry out of our project will help to develop effective strategies for the clinical treatment of nervous system degenerative disease. |
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