Effects Of EGCGon NSC34 Cells Stable Trasfected With TDP43

Neuroblastoma-spinal cord (NSC) was fused the aminopterin-sensitive neuroblastoma with motor neuron-enriched embryonic day 12-14 spinal cord cells. NSC-34 cell line not only overcome the shortage of primary motor neurons in culture and time-consuming, laborious, spending high enough, but also retaining the original features of primary motor neuron..NSC-34 is commonly used cell line to study amyotrophic lateral sclerosis(ALS).TDP43 is a ubiquitously expressed chromosome 1 encoded TAR DNA-binding protein with a molecular weight of 43 kDa .Until 2006,TDP43 (43-kDa TAR DNA-binding domain protein) was reported that was a major const- ituent of ubiquitin-positive cytoplasmic aggregates present in neurons of patients with fronto-temporal lobular dementia and ALS. Then , more and more people pay acttetion on the role of TDP43 in the degeneration disease .TDP43 is normally localized to the nucleus, however, in CNS tissue from patients who died of ALS, TDP43 is redistributed from the nucleus to the cytoplasm,where it appears to be distributed diffusely, or to aggregate as a component of ubiquitinated inclusions (UBIs). Recently,30 kinds of TDP43 mutations have been reported in both sALS and fALS cases. Evidence show that TARDBP genes, is likely to be a high ALS mutations gene following the SOD1 gene。In our study ,we want to observe the role of different TDP43 in NSC34 cell line and then explore the role of EGCG in TDP43 stable transfected cell line.PartⅠThe culture of NSC34 cells stable trasfected with TDP43 and the change of LDHObjective:To explore the culturing method of NSC34 cells stable trasfected with TDP43 and the change of LDH in the four cell lines .Thus,we will get the message of the reletionship between TDP43 and amytrophic lateral sclerosis ,which may be useful for the future study in the prevement and treatment .Methods:We used four different NSC34 cell lines that stabled transfected four different stable TDP43 plasmid. These four cell lines constructed by our laboratory. We used the methods that cultured the NSC34cell line to culture the four cell lines ,then bloted the four different TDP43 transfected NSC34 cells in the method of using high saturation antibiotics and selected the monoclonal cells. finally, the four cell lines was identificated by the fluorescence immunoassay assay.Firstly , we dislodged the stable transfection Empty vector, TDP43 Wide type, TDP43 Q331K and TDP43 M337V NSC34 cell line from the liquid nitrogen, used alcohol rub them three time , then shaked in 37℃water in order to reduce the time of the cell anabiosis .As soon as the cell anabiosis, removed them to the centrifuge tube that adding 10% serum containing DMEM medium. Secondly ,to centrifugate the cell and then discard the supernatant, add 10% serum containing DMEM medium, inoculate them in 25cm2one-time plastic flasks .Finally, we spined the bottle to the semi-loose state and put the cell in the incubator with 37℃, 5% CO2. Untill the cell grew 80%-90% confluence ,plated the four TDP43 NSC34 cell lines in 24-well cell culture with the density 10000cells/ hole. After 24 hours, we saw the cell in good condition by the inverted microscope. Then we changed the medium and waited 24 hours until the cells recovery and then collected the medium and cells for measue the levels LDH of the four cell lines.Results:We plated the four stable transfection cell lines in 250 thousand /blottle, they grew in 80% ~ 90% confluence after inoculated 2-3 days later. We saw cells were in good condition by the inverted microscope. Compared the four cell lines, we could see that the LDH level of TDP43 Wide type cell line the TDP43 Q331K cell line and the TDP43 M337Vcell line is higher than the Empty vector cell line, P <0.05, with statistical significance;the LDH lever of the stable transfected TDP43 Q331K were significantly higher than the other cell lines, P <0.05, with statistical significance.Conclusion: Compared with empty vector cell line the ,the level of LDH in the Wide type cell line is higher ,which was uniformity with the before report.The level of LDH of the stable transfected TDP43 Q331Kcell line is higher than TDP43 Widetype cell line, which indicated the function of these two genotypes of TDP43 is different. And this mutant TDP43 Q331K can produce a higher oxidative damage on the motor neuron-like cell line NSC34 cells. PartⅡEffects of EGCG on NSC34 cells stable trasfect with TDP43 Q331KObjective:To obersve the protective effects of EGCG on stable transfected with different TDP43 on NSC34 cell lines and explore the use of EGCG on the degeneration disease.Methods: We recoveried the TDP43 Q331K NSC34 cells and then put them in the incubator that in the 37℃, 5% CO2 condition . 24 hours later, we saw that the cells shape had been recoveried by the inverted microscope. Until cell growth reached 90% confluence, we plated the cells in 96-well cell culture plate, the density was 3000 / hole. 24 hours later, the cells were randomly divided normal control group and the EGCG intervention group that EGCG5μmol / L for 48 hours.48 hours later,we collected the all medium of two group cells in order to measure the lactate dehydrogenase (LDH) levels of the two group.Results:After 48 hours treated by EGCG in 5μmol/L the morphological changes of cells were not observed with invert microscope, compared with normal control group.The level of the LDH was difference. The LDH level of the cells that treated by EGCG 5μmol/L 48 hours was significantly decreased compared with non-interference.(P<0.01).Conclusion:These data indicated that EGCG could significantly reduce the cell LDH level, it may be useful for the cells to resistant the damage of lipid peroxidation that because of the mutant TDP-43. EGCG may be an effective agent for neurodegenration,which may play a role in prevention and treatment of the amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases.