The Role Of Notch Signalling Pathway On AD Cell Model

Objective: AD cell model was established on SH-SY5 Y cells which were treated by A?_25-35.The cell apoptosis and related signaling pathway genes were detected by different methods, the expression of TFF3 were also explored. Methods: 1.AD cell model was established using different concentrations of A?_25-35?10, 20, 40, 80?mol/L?, and the best treating time and concentrations of A?_25-35 were screened using cell survival rate by MTT method.2. Morphological characters of SH-SY5 Y cells were observed by H-E and Hoechst33258 staining under the microscope. Apoptosis of DNA fragments was detected by DNA ladder and changes of Caspase apoptosis related gene3, 8, 9 also explored in order to verify the efficiency of cell model. 3. Then mRNA and protein expression of Notch-1, Hes-1, TFF3 were detected by RT-PCR and Western blot. 4. Immunocytochemical staining was used to detect the location and changes of TFF3 protein. Results: 1. The cell survival rate was gradually decreased with the increased concentration and using time of A?_25-35, the optimal dosing concentration and time were screened at 20?mol/L, 48 h on SH-SY5 Y cells at last. 2. The cellular shapes of normal group were polygon or fusiform with uniform and blue stained nuclei, pinkish cytoplasm. In drug group, the cells were irregular with shrinked, dark blue nuclei and pale pinkish cytoplasm under the common microscope by H-E staining. 3. Hoechst 33258 staining results showed that the nuclei were observed with obvious nucleolus, emitting homogeneously blue fluorescence in the normal group, and typical morphological changes of apoptosis were happened with shrinked, dense nuclei gathered in groups, emitting enhanced blue fluorescence under the invert fluorescence microscope. Agarose gel electrophoresis showed obviously ladder-like DNA banding by DNA ladder. 4. RT-PCR results showed that the mRNA expression of caspase-3, 8, 9, Notch-1 and Hes-1were significantly higher in the drug group than that of the normal group. The mRNA expression of TFF3 was lower in the drug group than that of normal group. 5. Western blot results were consisted with the results of corresponding mRNA. 6. TFF3 immunocytochemical results showed that strong immunoreactive positive signal of TFF3 protein were located in cytoplasm with large, nucleolus in the central of cell in normal group, A?_25-35 drug group compared with normal control group, the cell shrinkage becomes circular or irregular shape. TFF3 immunoreactive signal was weakly in cytoplasm with shrinkage and dense nucleus in cytoplasm. Conclusion: 1. 20?mol/L A?_25-35 on SH-SY5 Y cells at 48 h, could induce AD cell model and made SH-SY5 cells apoptosis. 2. Notch signal pathway were activated, resulted in the decrease of TFF3 and apoptosis in A?_25-35 induced the AD cell model.