Effect Of Oxygen-Glucose Deprivation On Expression Of Cdh1and Its Role In Reactive Proliferation Of Astrocytes

ObjectiveThe pathological process of the reactive proliferation of astrocytes during the development of a variety of central nervous system diseases are very common. Combined effect of both the advantages and disadvantages of reactive proliferation of astrocytes are more inclined to harmful aspects to neurons, or even the entire central nervous system. To study the specific mechanisms of reactive astrocyte proliferation after cerebral ischemia and effectively inhibit the proliferation will be important and significance for the treatment of ischemic brain damage.Anaphase promoting complex(APC) and its regulatory subunit Cdhl could together degradation of cell cycle proteins to inhibit cell proliferation through the ubiquitination, while APC-Cdh1controls glycolysis though fructose-2,6bisphosphatase3(Pfkfb3), and glycolytic metabolism and cell proliferation are mutually reinforcing factors, on the other hand, after global cerebral ischemic injury, Cdh1protein was decreased in rat hippocampus, so we hypothesis that APC-Cdh1play an important role in the reactive proliferation of astrocytes, but the exactly mechanism is not clear. We put our experiments into practice by four steps.(1) To construct and indentify model of rat astrocytes reaction of the proliferation;(2) To explore the influence and mechanism in Cdhl protein expression of astrocytes during oxygen-glucose deprivation;(3) To construct and indentify recombinant lentivirus vector of rat Cdhl gene;(4) To explore the effect of Cdhl lentivirus on reactive astrocytes.Methods and Results(1) To construct and indentify model of rat astrocytes reaction of the proliferation Methods:Rat astrocytes was cultured and purified in vitro, and their purity are identified by IHCA. To construct confined and hypoxia little space for oxygen-glucose deprivation, and then to detect oxygen cotent and oxygen partial pressure of Earle's equilibrium liquid at different time(10min,30min,1h,3h,6h,9h) for evaluation the effect of oxygen-glucose deprivation; after conducting astrocytes with oxygen-glucose deprivation for1h and reoxygenation for48h, then detect proliferation by ambi-immunohistochemistry; after oxygen-glucose deprivation for3h,6h,9h, to detect change of cell cycle by PI Flow cytometry assay and detect expression of mRNA of Cdhl and of cell cycle protein B1of downstream substrate by Realtime-PCR.Results:The purity of astrocytes cultivated successfully in vitro>90%; Compared with control group, oxygen partial pressure in group of oxygen-glucose deprivation all descended(P<0.05); Compared with time of10min, the other time descended the same(P<0.05), there is no significantly difference between group of30min and1h,3h,6h,9h(P>0.05); Compared with control group, the processing of oxygen-glucose deprivation for1h and reoxygenation for48h would induce proliferation of astrocytes(P<0.05); oxygen-glucose deprivation for3h did not induce the proportion change of sell in S phase, proportion of sell in S phase in group6h and9h decreased obviously(P<0.05); oxygen-glucose deprivation for3h did not change Cdh1and CyclinB1mRNA level, expression of Cdh1mRNA descended and expression of CyclinB1mRNA in group6h increased, and compared with control group there were both statistical differences(P<0.05).(2) To explore the influence and mechanism in Cdh1protein expression of astrocytes during oxygen-glucose deprivationMethods:To culture purified cerebral cortex astrocytes of rats in vitro, randomly divided into the control group, oxygen-glucose deprivation (OGD) for1h and recovery group (group SD),6h-OGD and recovery group (group MD), Cdh1and Skp2protein expression was detected by Western blot; Astrocytes were randomized into control group,6h-OGD group (group D), hypoxia for6h group (group G), the expression of Cdh1protein was detected by Western blot, blood glucose meter was used to test glucose change.Results:Compared with control group, the expression of Cdh1in group SD and MD decreased, the expression of Skp2increased (P＜0.05); Compared with control group, the expression of Cdh1in group D significantly decresed, there were statistical differences between group D and G (P＜0.05); glucose uptake rates of extracellular fluid in group G was lower than control group (P<0.05).(3) To construct and indentify recombinant lentivirus vector of rat Cdh1geneMethods:The artificially synthesized full lenth DNA of rat Cdh1gene was inserted in the pGC-FU vector, which was isolated by restriction enzyme digest with Age I. The positive recombinant was identified by PCR analysis and sequence analysis. Six culture dishes of293T cell were randomly allocated into intervention group and control group. Expression of GFP protein was detected by fluorescence microscope and Western blot in pGC-FU-Cdh1transfected293T cells. Lentivirus pacaging, concentration and titering.Results:The PCR analysis and sequence analysis demonstrated that the size and position of Cdh1gene insertion were consistent with the design. Western blot analysis showed specific expression of protein of Cdhl-GFP in pGC-FU-Cdhl transfected293T cells, and have no expression in control group (P=0.014). Lentivirus titering by Real-time quantitative PCR was2×108TU/ml.(4) To explore the effect of Cdhl lentivirus on reactive astrocytesMethods:Rat astrocytes was cultured and purified in vitro, then randomly divided into Cdhl lentivirus group and control lentivirus group, the expression of green fluorescent protein was observed by fluorescence microscope, Cdhl and Skp2protein expression were detected by Western blot. And astrocytes were randomly divided into oxygen-glucose deprivation and recovery group, Cdhl lentivirus group and control lentivirus group, after1h of oxygen-glucose deprivation and recovery for48h, the proliferation of cell was detected by CCK-8and double-label immunohistochemical analyses.Results:Compared with control lentivirus group, the expression of Cdhl was increased, and Skp2was decreased (P<0.05). Compared with oxygen-glucose deprivation and recovery group, reactive astrocytes had been inhibited in Cdhl lentivirus group (P＜0.05), while control lentivirus group had no change (P>0.05)Conclusions(1) Mature astrocytes cultivated successfully were conducted oxygen-glucose deprivation, reached steady state after30min, woule last9h without difference, after oxygen-glucose deprivation for1h and reoxygenation for48h, induced proliferation of astrocyte; proliferation of astrocyte did not change level of Cdhl mRNA.(2) The expression of Cdhl proteins in astrocytes after OGD was decreased, and the change was related to the lack of glucose in extracellular fluid.(3) The effective recombinant lentivirus vector of rat Cdh1gene was successfully constructed and may serve as the basis for the further study of Cdhl gene function. (4) Cdh1lentivirus have effectively function on its downstream substrates and inhibitory action on reactive proliferation of astrocytes after OGD.