The Fragmentation Of Golgi Apparatus After Oxygen-glucose Deprivation Followed By Reperfusion And Its Underlying Mechanism

Chapter Ⅰ Morphological Alterations of Golgi Apparatus after Oxygen-glucose Deprivation Followed by ReperfusionObjective:To elucidate the morphological alterations of Golgi apparatus in mouse N2a cells upon oxygen-glucose deprivation followed by reperfusion (OGD/R), as well as the expression pattern of GM130protein.Methods:We employed OGD/R model to simulate ischemic-like conditions on N2a cells in vitro. Cellular viability and apoptosis were measured by CCK-8kit and fluorometric TUNEL test. The morphologic change of Golgi apparatus was determined by immunocytochemistry. The expressions of GM130and phospho-GM130were analyzed by Western blot.Results:1. In CCK-8test, OGD/R markedly reduced cellular viability in mouse N2a cells at6-hour,12-hour,24-hour and48-hour time points after OGD/R, compared with control group (P<0.01).2. In fluorometric TUNEL test, the proportion of apoptosis cell was measured. At0-hour and6-hour time points after OGD/R, the apoptosis rates showed no difference with control group (P>0.05), while the rates were gradually increased during reperfusion and reached a climax at the48-hour time points (P<0.05).3. In immunocytochemistry results, Golgi apparatus showed swelling and slightly fragmentation at24-hour time points after OGD/R, while it fragmented and scattered seriously at48-hour time points after OGD/R.4. Upon OGD/R, the expressions of GM130showed no significant differences compared with control group (P>0.05), except for48-hour time points after OGD/R (P<0.05). The expressions of phospho-GM130were little at0-hour and6-hour time points after OGD/R, as well as that of control group. But the levels of phopho-GM130were increased significantly at24-hour and48-hour time points after OGD/R (P<0.05).Conclusion:1. The fragmentation of Golgi apparatus is induced by OGD/R treatment in mouse N2a cells.2. OGD/R induces the phosphorylation of GM130, which is possibly related to the morphological alteration of Golgi apparatus. Chapter Ⅱ The Effects of Cdk1Inhibitor on the Morphology of Golgi Apparatus after OGD/RObjective:To elucidate the effects of GM130phosphorylation on the morphology of Golgi apparatus after OGD/R treatment by inhibiting the GM130phosphorylation with Cdk1inhibitor, Purvalanol A.Methods:The expressions of Cdk1protein and mRNA after OGD/R were tested by Western blot and Real time PCR. Making OGD/R treatment after intervening by Purvalanol A. Cellular viability and the morphology of Golgi apparatus were measured using the CCK-8kit and immunocytochemical method. The expression levels of GM130and phospho-GM130in cell were determined by immune fluorescence confocal techniques. The expression levels of Cdk1, GM130and phospho-GM130were further determined by Western blot with Cdk1inhibitor.Results:1. The levels of Cdk1protein and Cdk1mRNA were increased significantly at12-hour,24-hour and48-hour time points after OGD/R (P<0.05).2. We found a mild reduction of cellular viability on24-hour and48-hour time points after OGD/R with Purvalanol A treatment (P<0.05). 3. Immunocytochemistry results showed that the Golgi apparatus only underwent swelling and slightly fragmentation at48-hour time points after OGD/R with Purvalanol A treatment.4. By immune fluorescence confocal technique, we found that the distribution of phospho-GM130in cell was highly coincident with that of GM130. The expression of phospho-GM130elevated at12-hour,24-hour and48-hour time points after OGD/R without Purvalanol A treatment. By interfering with Purvalanol A, the expressions of phospho-GM130in N2a cells after OGD/R were at very low levels.5. In Western blot, the levels of Cdkl after OGD/R were inhibited by Purvalanol A treatment, as well as the expressions of phopho-GM130(P<0.05).Conclusion:1. The level of Cdkl is increased after OGD/R, while the inhibition of Cdkl down-regulates the phosphorylation of GM130.2. Cdkl inhibitor could alleviate the swelling and fragmentation of Golgi apparatus after OGD/R, possibly through the down-regulation of GM130phosphorylation.Chapter III The Effects of Cdkl Gene Silencing on the Morphology of Golgi Apparatus after OGD/RObjective: To construct the Cdkl targeting shRNA to inhibit the expression of Cdkl and to elucidate the effect of Cdkl on the morphology of Golgi apparatus after OGD/R treatment.Methods:We designed3shRNAs for Cdkl targeting, built a recombinant vector and transfected them to N2a cell. We observed the expressions of green fluorescent protein by fluorescent microscope, detected the expressions of Cdkl protein and mRNA, and then picked a most effective shRNA plasmid for inhibition. Taking positive-transfected recombinant plasmid Cdkl-shRNA-1as experiment group, negative-transfected recombinant plasmid Cdkl-shRNA NC as control group and untransfected plasmid as blank control group. After OGD/R, the morphology of Golgi apparatus in each group was observed by immunocytochemistry method and the expressions of Cdkl, GM130and phospho-GM130were detected by Western blot.Results:1. We designed3shRNAs for Cdkl targeting, connected them to hU6-GFP-SV40-Neomycin vector and identified them.2. Cdkl-shRNA-1transfected cells exhibited significant lower quantity in mRNA compared with Cdkl-shRNA-2group and Cdkl-shRNA-3group (P<0.05).3. Cdkl-shRNA-1transfected recombinant plasmid group showed a higher suppress rate compared with Cdkl-shRNA-2group and Cdkl-shRNA-3group (P<0.05). Negative-transfected recombinant plasmid group showed no difference compared with blank control group in the expressions of Cdkl protein (P>0.05).4. The cell combined with positive-transfected recombinant plasmid group (Cdkl-shRNA-1) showed no obvious fragmentation of Golgi apparatus after OGD/R.5. The cells combined with positive-transfected recombinant plasmid group (Cdkl-shRNA-1) showed the lower level of phospho-GM130protein after OGD/R compared with negative-transfected recombinant plasmid group and blank control group (P<0.05).Conclusions:1. Cdkl gene targeting shRNA vector is successfully constructed.2. Down-regulation of Cdkl gene alleviates the fragmentation of Golgi apparatus after OGD/R, possibly related to the decreased phosphorylation of GM130.