Pilot Study Of The Expression Of Atrial Natriuretic Peptide In The Vitreous Humor And Epiretinal Membrane Of The Patients With Proliferative Diabetic Retinopathy

Object:To determine the expression of atrial matriuretic peptide(ANP) in the vitreous humor and epiretinal membrane(ERM) of the patients with proliferative diabetic retinopathy(PDR) and evaluate the possibility that ANP could be involved in the process of PDR.And to establish the proach of study of ERM.Methods:Part one:Ten cases of ERM samples from PDR patients(test group) and five cases from patients with proliferative vitreoretinopathy(PVR)(control group) were collected.The expression of ANP and the characteristic of proliferation of two groups were determined by immunohistochemistry.The mean ANP ratio of two groups was analyzed by Mann-Whitney U test.Part two:Thirty cases of vitreous humor samples from PDR patients(test group)and non-PDR(control group) patients,respectively,were collected.ANP was determined by radioimmunoassay(RIA) and the protein content was determined by the method of Bradford.ANP concentrations,protein concentrations and ANP/protein ratios were analysed between two groups by Mann-Whitney U test.Part three:Fifteen cases of ERM samples from PDR patients(test group)and non-PDR(control group) patients,respectively,were collected.The expression of ANP gene was determined by semiquantitative reserve transcription-polymerase chain reaction(RT-PCR) The resulting ANP amount was devided by the GAPDH amount to obtain a normalized value.The final value was analysed between two groups by Mann-Whitney U test.Results:Part one:Membranes from both groups consisted of different types of cells as shown in Hematoxylin-eosin staining.Immunohistochemistry studies have demonstrated these membranes mainly consist of glial cells and RPE cells,while PDR membranes also had vascular endothelial cells(EC).ANP is present in both glial cells and RPE cells,and in vascular EC in PDR membranes.The mean ANP ratio in test group and control group is 32.1%(range:0～52.0%,median:33.5%) and 1.6%(range:0～8.0%,median:0%),respectively(P＜0.01).Mean GFAP and CK ratio in test and control group were 56.4%(range:52.1～60.3%,median: 56.5%),21.5%(range:18.3～24.6%,median:23.3%);58.2%(range:55.1～60.9%,median:58.4%)和22.7%(range:19.7～25.6%,median:22.2%), respectively(P＞0.05).Part two:Mean ANP concentrations in test group and control group were(68.23±16.65)pg/ml(range:40.11～99.56pg/ml, median:66.30pg/ml) and(38.45±7.62)pg/ml(range:30.10～57.32pg/ml, median:36.86pg/ml),respectively(P＜0.01).Mean protein concentrations were (810.53±188.60)ug/ml(range,401.33～1361.70ug/ml,median: 786.58ug/ml)and(596.17±161.80)ug/ml(range:375.69～1136.37ug/ml,median: 570.04ug/ml),respectively(P＜0.01).Mean ANP/Protein ratio were(0.09±0.02)(rang,0.05～0.16,median,0.09)and(0.07±0.02)(range,0.04～0.14, median,0.06),respectively(P＜0.01).Part three:Mean peak values of ANP and GAPDH in test group were 188.5±53.6(range:109.3～255.0,median:254.0) and 237.9±31.1(range:162.2～255.0,median:254),respectively.Their mean ratio is 0.8±0.3(range:0.51～1.4,median:0.8).Mean peak values of ANP and GAPDH in control group were 100.4±12.0(range:79.2～122.0,median:98.3) and 231.6±36.3(range:113.8～255.0,median:246.8).Their mean ratio is 0.4±0.1(range:0.31～0.82,median:0.4).The difference between two groups has statistical significance(P＜0.01).Conclusion:The exrpression level of ANP in PDR membranes and vitreous humor was significantly higher than that of non-PDR patients through immunohistochemistry,semiquantitative RT-PCR and RIA.Our findings suggest a role for ANP in PDR.RT-PCR technique is a reliable proach for ERM study.