The Study Of Expression On HnRNP A2/B1 After Ischemia-reperfusion Insults

Objective To develop an oxygen and glucose deprivation (OGD) model to mimic focal ischemia insults in PC 12 cells. After PC 12 cells were exposed to oxygen and glucose deprivation, the translocation of heterogeneous nuclear ribonulcleoprotein A2/B1 (hnRNP A2/B1) from the nucleus to the cytoplasm was examined. To identify whether hnRNP A2 and/or hnRNP B1 was translocated from the nucleus to the cytoplasm in rat cerebral cortex after Cerebral Ischemia/Reperfusion insults (Ischemia: the middle cerebral artery occlusion/reperfusion, MCAO/R), the respective polyclonal antibodies were also developed. And both of these polyclonal antibodies can be used for follow study of mechanism on hnRNP A2 and hnRNP B1.Methods Reconstruction OGD model according to the datas. PC 12 cells were exposed to OGD stress for different times (5 min,10 min,15 min,30 min,45 min,1 h, 3 h,6 h,12 h,24 h,48 h). The cells viability was measured by MTT assay. Additionally, the effects of deprivation of oxygen or serum or glucose on cells viability were assayed by MTT.Cells were exposed to deprivation of OGD, normal cultivation after oxygen deprivation and glucose deprivation, then measured whether the hnRNP A2/B1 translocated from the nucleus to the cytoplasm by immunocytochemistry and immunofluorescence methods in PC 12 Cells or neurons. Sodium sulfite with different concentrations of 0.5 g/L,1.0 g/L,2.0 g/L,5.0g/L instead of oxygen deprivation to treated cells for 4h,6h, then normal cultivation for 24h and measured the cells viability by MTT assay. In addition, the sodium sulfite was used to develop the model of OGD and make sure the localization of hnRNP A2/B1 in PC12 cells.The hnRNP A2 and hnRNP B1 specific oligopeptide were coupling on the carrier protein KLH which were used as antigen to immunize New Zealand rabbits. We employed immunohistochemistry to examine the expression of these two anti-serum in rat cerebral cortex after Cerebral Ischemia/Reperfusion insults.Results OGD model was established in PC 12 cells successfully. The LD50 of PC 12 cells with OGD was determined to be 10.3 min. The glucose but not oxygen or serum played a significant role in the survival of PC 12 cells.After OGD for 24h, hnRNP A2/B1 was significantly gathered to perinuclear region and in the nucleus showed the phenomenon of vacancies, other experiments hadn't this phenomenon. Instead of oxygen deprivation the result of using sodium sulfite was similar and comparable, and the 5g/L is the better concentration similar with oxygen deprivation. When using the sodium sulfite with concentration of 5g/L instead of oxygen deprivation to OGD for 24h, hnRNP A2/B1 has a slight aggregation phenomenon.The hnRNP A2 and hnRNP B1 specific oligopeptide were coupling on the carrier protein KLH which were used as antigen to immunize New Zealand rabbits and got the anti-serum. Using the dilution ratio of 1:500, we can clearly observe the sublocation of hnRNP A2 or hnRNP B1.Conclusion We successfully developed the OGD model in PC 12 cells, which provides useful references for revealing permanent focal ischemia insults and relative illness. Although the hnRNP A2/B1 proteins mainly localized in the nucleus area, after OGD for 24h it significantly aggregated in the perinuclear. Provides useful references for revealing permanent focal ischemia insults and relative illness.