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The Expression of P-glycoprotein in the Neurons Injured by the Oxygen and Glucose Deprivation and the Effects of P-glycoprotein on the Damage of Neuron

Author: LiShiXu
Tutor: GuanYangTai
School: Second Military Medical University
Course: Neurology
Keywords: P-glycoprotein cerebral cortical neurons oxygen and glucose deprivationapoptosis inhibitor
CLC: R743.3
Type: Master's thesis
Year: 2013
Downloads: 22
Quote: 0
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Abstract


Neuronal ischemia-reperfusion injury is an important pathophysiological process ofacute ischemic stroke, which involves oxidative stress, inflammation, calcium overload,excitatory amino acid toxic effects. It is the focus of current research in this field byexploring the protection mechanism of neuronal ischemia-reperfusion injury to providenew ideas for clinical treatment. P-glycoprotein belongs to the ATP-binding cassettetransporter protein superfamily. P-glycoprotein, key function as media by dischargingsubstance or drug to avoid the abnormal substances accumulated in the cells, is mainlylocalized to the luminal (blood side) plasma membrane of the blood-brain barrier capillaryendothelial cells in the central nervous system. In cerebral ischemia, epilepsy and otherdiseases, P-glycoprotein expression is up-regulated in neurons, glial cells. However, themechanism and pathophysiological significance of the up-regulated expression remainsunclear. Therefore, the purpose of this study is to investigate the change in expressionlevels of P-glycoprotein in neurons after ischemia-reperfusion injury and its impact onneuronal damage.Part Ⅰ The expression of P-glycoprotein in the neurons injured by theoxygen and glucose deprivationObjective:To value the expression level of P-glycoprotein from those neurons which weredeprived oxygen and glucose.Methods:Fostered the primeval generation neurons from cerebral cortex of mice in vitro, andthen use these neurons to build an oxygen and glucose deprived and oxygen and glucoseregenerated or recovered (OGD/R) model. All the neurons of this experiment were dividedinto4groups based on the different length of culture time after the regeneration of oxygenand glucose: control group, OGD/R model in6hours group (6hours after recover oxygenand glucose after deprivation), OGD/R model in12hours group (12hours after recoveroxygen and glucose after deprivation), and OGD/R model in24hours group (24hoursafter recover oxygen and glucose after deprivation). Firstly, use method of MTT toestimate the tendency of the change of vitality of neurons from different groups. Second,use method of real-time quantitative PCR to examine the average value of relative expression of ABCB1a、ABCB1b mRNA in the neurons from different groups. And duringthe third step, we used immunofluorescence method to mark the monoclonal antibody ofP-glycoprotein, and then observed whether in the neurons exist the expressions ofP-glycoprotein. The change of average value of the fluorescence strength which stands bythe quantity of P-glycoprotein also was observed.Results:We cultured amount number of primeval generation cerebral cortex neurons of micein vitro, and built the OGD/R model of primeval generation cerebral cortex neuronssuccessfully. Method of MTT indicated that：the vitality of neurons declined successivelyby the length of deprived oxygen and glucose. Method of real-time quantitative PCRshows group OGD/R model in12hours occupies the highest average value of relativeexpression of ABCB1b mRNA. There is no obvious difference of average value of relativeexpression of ABCB1b mRNA between group OGD/R model in6hours and group OGD/Rmodel in24hours. There is no group can found the expression of ABCB1a mRNA.Immunocytochemistry method indicated that notable green immunofluorescence mark canbe determined from every group except control group. Group OGD/R model in12hoursowns the most significant quantity of fluorescence mark while there is nearly no markercan be found in control group. This is interesting; group OGD/R model in24hours haslower average value of the fluorescence strength than group OGD/R model in12hours.Conclusion:1. OGD/R model can be used for the research of P-glycoprotein.2. The level of mRNA and protein of P-glycoprotein are highly expressed afterneurons injured by deprive oxygen and glucose. The level of mRNA and protein ofP-glycoprotein increased most highly when neurons were deprived oxygen and glucose in12hours. Our study also shows that the expression level of P-glycoprotein may relate tothe length of time after the neuron regenerated by oxygen and glucose.Part Ⅱ The effects of P-glycoprotein in neurons of OGD/R model on thedamage of neuronsObjective:To determine the relationship among the change of the expression value ofP-glycoprotein and the extent of apoptosis of neuron、Bax and Caspase-3. Methods:Based on the modeling of OGD/R of neuron, we used the3rdgeneration ofP-glycoprotein inhibitor-Tariquidar（TQD）as process mode. First of all, we use themethod of MTT to choose the suitable operating purity of TQD. This study subgroup into3groups: control group, group OGD/R which included neurons of OGD/R model, groupTQD+OGD/R which included neurons of OGD/R model and then processed by TQD.Secondly, we use method of real-time quantitative PCR to examine the average value ofrelative expression of ABCB1b、Bcl-2、Bax、Caspase-3mRNA of the neurons from eachgroup. Also immunocytochemistrical method was adopted to mark P-glycoprotein andprotein Bcl-2、Bax、Caspase-3aimed to evaluate their expression and average fluorescentstrength in different group. At the same time, we chose Western Blot method to analyze theexpression extent of P-glycoprotein and protein Bcl-2、Bax、Caspase-3. Last, doublestandard flow cytometer of AnnexinⅤ/PI was used to examine the apoptosis extent ofneurons from every group.Results:1. Changes of expression of P-glycoprotein: after inhibited by TQD, the amount ofexpressed P-glycoprotein from protein is declined while the expression of P-glycoproteinfrom mRNA is keep stable.2. Changes of expression of Bcl-2and Bax: pro-processed by TQD，the expression ofBcl-2in the neuron of OGD/R model is decreased while the expression of Bax andCaspase in the same situation is increased.3. Changes of expression of Caspase: after the pro-process of TQD, the number ofexpressed Caspase raised than it found in other group without pro-processed. And we alsofound the deterioration of neuron is worse than it from other group.4. Double standard flow cytometer of AnnexinⅤ/PI analysis: the number of deadneurons is increased due to the usage of TQD.Conclusion:1. TQD can inhibit the expression of P-glycoprotein effective.2. After P-glycoprotein inhibited by TQD, the extent of damage of neuron willbecome more serious.3. P-glycoprotein may play an important role of protection for ischemic neuronsuffers the injury caused by re-perfusion.

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CLC: > Medicine, health > Neurology and psychiatry > Neurology > Cerebrovascular disease > Acute cerebrovascular disease ( stroke)
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