Effect Of Glucose And Hypoxia On AQP4 Expression Of Retina Mǔller Cell Culture In Vitro

Diabetic retinopathy (DR) is the common and serious microvescularcomplication in diabetes,and is one of the four main causes of blindness.So clarifying the pathogenesis mechanisms of DR and questing for validprevention and cure method are very important to people.Retina MǔllerCell is the specialized glial cell, and expresses pressure-activated channeland neural transferred receptor. It regulates neural activity by adjustingextracellular neural active matter ,such as K+,glutamic acid,H+. It has thephysiological effect of keeping retina natural structure,nourishing retinaneuron and participating in the barrier of blood-retina. And it is concernedwith retina pathology. Mǔller Cell, though whole retina from innerboundary membrane to outer boundary membrane, is liable to pathologicalfactor. The investigation approved, Mǔller Cell is an important role in thepathogenesis of DR by excrete multigrowth factors, for example bFGF,VEGF,IGF-1,TGF-β.Water transportion is the basis of cellular survivor. AQPs is theimportant physiological factor of regulating water osmosis and signaltransduction. AQP4, expressed in retina Mǔller Cell, participates inpreserving excitability during retina signal transduction and influencinghydration by adjusting retina water permeability. Based on thecolocalization of Kir4·1 potassium channels and AQP4 in specificmembrane domains of retina Mǔller cells, it was proposed that AQP4participate in K+ siphoning and regulate water permeability with Kir4·1potassium Channels. Therefore, investigating the expression change ofAQP4 in retina Mǔller Cell on the condition of diabetes, pathogenesis ofAQP4 on DR and studying the new medication to protection of DR are veryimportant.This experiment we adopted the method of tissue suspended curture toestablish primary culture of rabbit retina Mǔller Cell.Then we applied themethod of immunofluorescence stain techniques,flow eytophotometer andRT-PCR investigate the effect of glucose concen tration of 30,40,50mmol/L and hypoxia of 3,12,24,48 hours on AQP4 expression of retinaMǔller Cell. And we adopted AO/EB stain to detect apoptotic Mǔller Cellunder different hypoxia time. Our results are as follows:1,According to the result of phase contrast microscope, HE stain,immunocytochemical stain and transmission electron technique, the purityof Mǔller Cell was more than 95%. Mǔller Cell had the characteristic oflong shuttle,growth slowing, overlapping row for noncontract inhibition,GFAP antibody staining positive, and Ⅷ facter correlated antigen antibodystaining negative. Its nucleus was big and anomalous,and had two or moremucleoli , ample cytoplast by electron microscope. Besides Mǔller Cellhad rough endoplasmic reticulum , mitocho-ndrion , Golgi complex,free ribosome, complex, free ribosome,microfilament, and haddiagnostic intermediate silk (8～10 nm).2,The result of immunoflurecense revealed the AQP4 expression ofretina Mǔller Cells was positive and green flurecense was the stronger atthe natural control group. The AQP4 expression of Mǔller Cells decreasedobviously at the high glucose group(P<0.01).Besides,with concentrationof glucose increasing and time longer, the decreased extent of AQP4expression also increased gradually. On the condition of hypoxia, theAQP4 expression of Mǔller Cells decreased obviously(P<0.01). Withhypoxia time longer , the decreased extent of AQP4 expression in MǔllerCells also increased(P<0.01). At 24h of hypoxia, grey value is 159.8±7.1,which is the lowest. At 48h of hypoxia, grey value is 162.7±0.6 and thedecreased extent of AQP4 expression has come back.3,The result of FACS indicated the locality of flurecense intensity is950 at the natural control group,which reveals AQP4 has the strongerexpression in Mǔller Cell. The position of wave crest advanced obviously atthe high glucose and hypoxia group,which suggest AQP4 expression inMǔller Cells decreased. With concentration of glucose increasing and timelonger, the position of wave crest advanced gradually.The position ofwave crest is 380 after 24h of hypoxia;the locality of wave crest advancedobviously compared to the natural control group(P<0.01).But on 48h ofhypoxia the locality of flurecense intensity is 477 and it indicates thedecreased extent of AQP4 expression has come back.4, RT-PCR revealed ,on the condition of 50mmol/l glucose of 3dand 24h of hypoxia ,AQP4 mRNA expression decreased obvi-ously.5, when Mǔller Cell was cultured at hypoxia condition, the resuleof AO/EB stain revealed the a majority of cells has a natural configurationat the natural control group and express yellow flurecense;Individualcellular configuration appears change after 3h of hypoxia,and chromatinhas orange flurecense.These results suggest early apoptotic cells appear.With hypoxia time longer , apoptotic Mǔller Cells were increasedgradually.However, within 48h of hypoxia, apoptotic Mǔller cells'number had not an prominent increased.On the condition of 72h ofhypoxia, cellular apoptotic ratio arrived at 23%,which indicated apoptoticcells' number increase obviously when time of hypoxia exceeds to 48h.These results suggested that glucose concentration and hypoxia effectobviously AQP4 expression of retina Mǔller Cells. They can make AQP4expression decreased and have time and concentration -dependent manner.But on the condition of 48h of hypoxia ,the AQP4 expressed decrease hascome back. Within 48h of hypoxia apoptotic Mǔller cells' number had notan prominent increased,which has no evident effect on the results. In theword, AQP4 participates in the development of DR by many approachs,and AQP4 expressed decrease is an protective response in organismforepart. This experiment provides a further investigation of pathogenesisof AQP4 on DR,and also provideds a theoretical and experimental basisfor prevention and cure of AQP4 in diabetic retinopathy.