Protective Effects Of 78kDa Glucose Regulated Protein Overexpression On Retinal Ischemia Reperfusion Injury

PrefaceRetinal ischemia reperfusion(RIR)injury is a frequent clicical entity.It often lead to serious visual impairment,yet there is no specific treatment.Therefore to find new ways to control RIR injury is imperative.Gene therapy is a new technique commence from 1990s opening up a new way of modern therapy.The pathophysiological mechanism of RIR injury is very complex and many factors are stress-related.Thus,intervention from the cells stress level may provide new idea for prevention or treatment of such diseases.78 kDa glucose regulated protein(grp78)is a major cytoplasmic chaperone.It plays important roles in protein quality control,signal transduction,proliferation,and cell death,and confer cytoprotection and assure survival after environmental stress.The protective function of the grp78 suggests that their induction could be an adaptive response evolved in mammals to protect cells against stress-induced cell death and be beneficial in situnations involving tissue or organ damage.Recently,overexpression of grp78 were reported to protect cells against cell death.Thus,the induction of grp78 opens up new therapeutic approaches to these diseases.We employed plasmid pEGFP-N1 as the vector to transfect grp78 into RPE cells and rat retina.We intended to find the role of grp78 gene in the protection of retinal cells against ischemia reperfusion injury.Objective1.To observe the expression and location of grp78 in the rat retina induced by ischemic reperfusion injury(RIR),and to assess the relationship between grp78 and retinal cell apoptosis.2.To construct the pEGFP-grp78 fusion protein eukaryotic expression vector and to detect their expression in human retinal pigment epithelium(RPE)cell lines.3.To investigate the effects of C₀Cl₂ induced hypoxia and H₂O₂ induced oxidative stress on RPE cells proliferation and the expression of grp78.4.TO investigate the transduction ability of PEGFP-grp78 into human RPE cells,and the protective effects on C₀Cl₂-induced hypoxia,H₂O₂-induced oxidative stress injury in these cells.5.To observe the expression of the grp78 after pEGFP- grp78 was transtected into the retina of the rats using ationic lipostme;and to investigate the protective effects of grp78 gene transferred on retinal ischemia reperfusion injury.MethodsPart1:Expression of grp78 in the rat retina induced by ischemic reperfusion injury and its relationship with cell apoptosis.Thirty-six wistar rats were equally divided into 6 groups randomly:normal control group and ischemia reperfusion groups(6,12, 24,48,72 hours after ischemia reperfusion).Each group included 6 rats.The rat retinal ischemia reperfusion model was established by acute elevated intraocular pressure through normal saline intracameral perfusion(110 mmHg×60 min).The expression of grp78 in rats retina was detected by immuno-histochemistry and semiquantitative RT-PCR methods at the different time points.Apoptosis was assessed by Tunel method.The results were analyzed by SPSS software,take the average optical density to perform statistical analyses.Part2:Construction of human grp78 eukaryotic expression vector and expression in RPE cells.1.The encoding fragment of human grp78 gene was obtained from a recombinant plasmid POTB7-grp78 using PCR,and it was digested by restriction enzymes Hindâ…¢and kpnâ… .Then the purified grp78 gene fragment was inserted into the GFP expression vector PEGFP-N1,and the recombinant plasmid PEGFP-grp78 was identified by PCR analysis and DNA sequencing.2.The recombinant plasmids were transfected into RPE cells by lipofectamin in method.RPE cell lines whith grp78 stable expression were established by G418 selection.The expression of grp78 gene was detected by RT-PCR;The expression fusion proteins were assayed by fluorescence microscopy.Part3:Effects of C₀Cl₂ and H₂O₂ on the expression of grp78 and proliferation of RPE cells.1.Human RPE cells was cultured in vitro.The C₀Cl₂ groups were exposed respectively in 100,200,400,800μmol/L C₀Cl₂ for 12 hours. The H₂O₂ groups were exposed respectively in 100,200,400,800μmol/L H₂O₂ for 12 hours;And cells without treatment were used as control. The technique of MTT was performed to evaluate the effects of cell proliferation.2.The expression of grp78 were explored by using immunocytochemistry at 8,12,16 hour points after C₀Cl₂(200μmol/L)or H₂O₂(400μmol/L)treatment.Part 4:The protective effect of grp78 gene transfection on RPE cells against hypoxia and oxidative stress.1.RPE cells were divided into 5 groups:â‘ The control group;â‘¡The RPE cells with C₀Cl₂(200μmol/L)treatment;â‘¢The RPE cells carrying recombinant grp78 gene with C₀Cl₂(200μmol/L)treatment;â‘£The RPE cells with H₂O₂(400μmol/L)treatment;⑤The RPE cells carrying recombinant grp78 gene with H₂O₂(400μmol/L)treatment.2.The viability of the cells were measured by the MTT method.Part5:Protective effects of grp78 gene transferred on retinal ischemia reperfusion injury in rats1.Cationic liposome containing pEGFP- grp78 gene was injected into the vitreal chamber.The expression of grp78 mRNA in rat retina was assessed by reverse transcribed polymerase chain reaction at the different time points(1d,1W,2W,4W)after transfected.2.Retinal ischemia was induced by increasing the intraocular pressure in rats.3.ERG was used to observe the retinal function.Apoptosis was assessed by TUNEL method. ResultsPart 1:1.The expression of grp78 was located in retinal ganglion cell layer and inner nuclear layer.After retinal ischemia reperfusion,the expression of grp78 was increased.The highest level of expression was reached at 24 hours after reperfusion.The level then began to decrease at 48 hours.2.No apoptosis cells were observed in the retina of normal rats.There was a number of TUNEL positive cells in the 6th～24th hour after ischemia-reperfusion follewed by a decrease at the 48th hour with the maximum at the 24th.3.The grp78 protein expression followed a similar pattern with Tunel detection.Part 2:1.A abuout 2000 bp fragment was obstained by PCR clone.We successfully constructed the expressing vector of pEGFP-grp78 fusion protein and the sequence results were matching to the cDNA of human grp78,which accession of the Genebank was NM-005347.2.RT-PCR showed that grp78 gene was overexpressed for at least 72h,which was detected 24h after pEGFP-grp78 plasmids were transfected into RPE cells.Green fluorescence was observed by fluorescence microscopy on the cells transfected with pEGFP-grp78 plasmids.Part 3:Proliferation of hRPE was decreased significantly by C₀Cl₂,H₂O₂ treatment.C₀Cl₂,H₂O₂ treatment up-regulate the expression of grp78 in human RPE cells in a time dependent manner. Part4:After C₀Cl₂,H₂O₂ administration,cell viability was significantly reduced compared with control group;While the viability of cells which were transfection with recombinant vector carrying grp78 gene were significantly higher than normal RPE cells.Part 5:1.One day after transfected gene grp78-pEGFP into rat eyes,the expression of grp78 mRNA was observed in the retina using RT-PCR and maintained stable on the forth week.2.Overexpression of grp78 could protect the rat retina against ischemia reperfusion injury.Conclusion1.Retinal ischemic reperfusion induces cell apoptosis in the retina.Grp78 may play important roles in the process.2.The eukaryotic expression vector PEGFP-grp78 was successfully constructed and expressed in the retinal pigment epithelium cell lines.3.Grp78 may have a protective effect on human RPE cells,the change of grp78 mRNA and the RPE proliferation is the molecular basis of the antiapoptosis of the RPE cells.The grp78 may play an important role in the development4.Overexpression of grp78 could protect RPE cells against oxdative and hypoxia injury,the probable mechainsms maybe concered with the inhibition of cell apoptosis.5.Cationic liposome can transfect Grp78-pEGFP gene into the retina of the rat effectively,and their expression can maintain 4 weeks after transfection.Overexpression of grp78 could protect the rat retina against ischemia reperfusion injury.