The Expression Of MafK And JDP2 In NSC-34 Cells Transfected With Mutant TDP-43 And The Effect On Nrf2/ARE Pathway

PART 1 CONSTRUCTION OF ALS MODEL STABLY EXPRESSING PDEST30-EGFP-TDP-43 PROTEINObjective To establish a stable NSC-34 cell line expressing EGFP-TDP-43.Methods First,build three kinds of eukaryotic expression vector,they are p DEST30-EGFP,p DEST30-EGFP-TDP-43-WT and p DEST30-EGFP-TDP-43-M337 V.Second,the three plasmids were introduced into NSC-34 cells via transfection techniques.Third,we use G418 to screen out stable expressing cell lines.At last we detect wheather p DEST30-EGFP,p DEST30-EGFP-TDP-43-WT and p DEST30-EGFP-TDP-43-M337 V expressed in Eukaryotic cells by using fluorescence microscope,RT-PCR and Western blots methods.Also we use MDA kit to detect the expression product of cell oxidative stress process in three stable expression cell lines.Results We successfully get the three kinds of stable expression cell lines after transfection and repeatedly screening by G418,they are p DEST30-EGFP-NSC-34 cell line,p DEST30-EGFP-TDP-43-WT-NSC-34 cell line and p DEST30-EGFP-TDP-43-M337VNSC-34 cell line.Under inverted fluorescence microscope,we observe the expression of green fluorescence are more than 95%.RT-PCR and Western blots results also confirms that only the wild group and mutation group express exogenous TDP-43 in gene expression levels and protein levels.MDA determination within the three groups of cells showed the oxidative stress level in mutation group were significantly higher than the empty sublet group and wild group(P<0.01,n=5),Yet,the empty sublet group and the wild group's oxidative stress level have little difference,and the difference have no statistically significance.Conclusions With p DEST30-EGFP,p DEST30-EGFP-TDP-43-WT,p DEST30-EGFP-TDP-43-M 337 V three plasmids transfected into NSC-34 cells selected by G418,we can successfully construct EGFP-TDP-43 cell lines stably expressing the protein,and thus we can provide useful cell experiments model for ALS subsequent functional studies.PART 2 THE EXPRESSION OF MAFK AND JDP2 IN NSC-34 CELLS TRANSFECTED WITH MUTANT TDP-43 AND THE EFFECT ON NRF2/ARE PATHWAYObjective To discuss the Nrf2/ ARE pathway's damage level in the mutant TDP-43 transfection of NSC-34 cell model;To observe Maf K protein and JDP2 protein's expression level and discuss their impact on Nrf2/ARE pathway in three cell models,thus we may provide the basis mechanism for the further study of ALS pathogenesis.Methods 1.The introduction of ALS cell models.The ALS cell models were subcultured in part 1.They are p DEST30-EGFP-NSC-34 cell line,p DEST30-EGFP-TDP-43-WT-NSC-34 cell line and p DEST30-EGFP-TDP-43-M337V-NSC-34 cell line.2.Set three groups,including the empty transformation group,the wild-type and the mutant group,seperatelly do the following.1)Use Western blots and electrophoresis to detect the total change of Nrf2 and the downstream phase ? detoxifying enzymes such as NQO1's expression level in each group cells;And detect Nrf2's expression level in cytoplasm and within the nucleus.2)Use malondialdehyde(MDA)kit to detect the level of oxidative stress.3)Use immunohistochemistry kit to observe cells' morphological changes.4)Use MTT assay kit to observe changes of cell apoptosis and cell activity.3.To detect the expression of Mafk protein in three groups.We use Western blots and electrophoresis techniques to detect Mafk protein's expression.4.To detect the expression of JDP2 protein in three groups.We also use Western blots and electrophoresis techniques to detect JDP2 protein's expressionResults 1.We have already built stably expressing p DEST30-GFP,p DEST30-GFP-TDP-43-WT,p DEST30-EGFP-TDP-43-M337 V three proteins' cell lines,they are the empty transformation group,the wild-type and the mutant group.2.Immunohistochemical results show that the mutant group's cell body are round and their protrusion are reduced and more shorter than the other two groups..3.MDA test results show that: MDA levels of mutant group cells are significantly higher than in the empty transformation group and the wild-type group(P<0.05),whereas no significant difference between the empty transformation group and the wild-type group(P> 0.05).4.PCR and Western blots results show that: In the mutation group,the total expression of Nrf2 are deceased and the Nrf2 expressed in the nuclear were accumulated.But the downstream effectors such as NQO1's expression was reduced,compared with the empty transformation group and the wild-type group,the difference have the statistically significance(P<0.05).5.MTT result show that:In the mutant group,the OD level was significantly lower than the empty transformation group and the wild-type group,the difference have the statistically significance(P<0.001),while no significant difference between the empty transformation group and the wild-type group(P>0.05).6.Under Western blots results level,the expression of Maf K were significantly decreased in mutant cells compared with the empty transformation group and the wild-type group cells,the difference have the statistically significance(P<0.05).7.Under Western blots result level,the expression of JDP2 were significantly decreased in mutant cells compared with the empty transformation group and the wild-type group cells,the difference have the statistically significance(P<0.05).Conclusions By using the newly build mutant TDP-43 transfected NSC-34 cell model,we find that the cellular antioxidant levels to reduce stress was damaged;The expression of Nrf2/ARE pathway's downstream effect factor were deceased while Nrf2 within the nucleus were accumulated;We also find that the two cis element of Nrf2/ARE pathway--Maf K and JDP2,participating in the antioxidant stress reaction,have the different protein express quantities between normal cells and mutant cells.Separatelly,Maf K expression and JDP2 expression both reduced in the mutant group cells.Oxidative stress damage may be associated with the two protein's expression change,it remains to be further studied.