| Diabetic retinopathy(DR)is a common microvascular complication of diabetes mellitus,of which the proliferative diabetic retinopathy(PDR)is one of the main blinding diseases in ophthalmology.The pathogenesis of DR is complicated,which many molecular mechanisms are involved in it.The anti-angiogenesis has become the key point in the study of DR since the formation of neovascularization is the most essential pathological feature of PDR.In addition,semaphorin,also known as axon guidance molecule,is a large family of signal proteins in secretory or cell membrane binding forms,widely expressed in many tissues such as nervous system,immune system,cardiovascular system and so on,among which semaphorin4D(Sema4D)is a crucial member of Semaphorin Ⅳ subfamily.In recent years,abnormal high expression of Sema4 D has been found in many tumor tissue neovascularization studies,and it has been found that Sema4 D can induce the proliferation of neovascularization by binding to high affinity NGF receptor plexus protein B1(plexin-B1).However,the related study of the expression and significance of Sema4 D in the eyes of PDR patients are rare at home and abroad.In this study,the author first studied the difference of Sema4 D expression in vitreous of PDR patientsand non-PDR patients.Then,the human retinal pigment epithelium(hRPE)was selected as the research object,and the expression of related indexes was detected by establishing a high glucose induced cell model through culturing the RPE cells in vitro.Furthermore,RNA interference was applied to silence the expression of Sema4 D gene to investigate the effect of Sema4D/plexin-B1 on the expression of VEGF and MMP-9 in high glucose cultured RPE cells,and to analyze the role of Sema4D/plexin-B1 in diabetic retinopathy and its possible mechanism.Part I Expression of Sema4 D and MMP-9 in vitreous of patients with diabetic retinopathy Objective:To study the expression of Sema4 D in the vitreous of PDR patients and non-PDR patients,and to discuss the possible role of Sema4 D in the development of PDR.Method:1.Clinical data:40 patients with proliferative diabetic retinopathy were selected as experimental group.Among them,20 patients who received intravitreal injection of anti-VEGF drugs before vitrectomy were divided into Group A.The other 20 patients underwent direct vitrectomy were divided into Group B.In addition,20 patients with macular holes with no history of diabetes mellitus were selected as control group.Clinical data such as age and gender were recorded.All patients were undergone the routine ophthalmologic examination,and the vitreous specimens were collected during operation.2.The content of Sema4 D,MMP-9 and VEGF in vitreous of patients in each group was detected by ELISA.3.The expression content difference in each group was compared by the statistical analysis.Result:1.The concentration of Sema4D(ng/ml)in vitreous of experimental groups,Group A,Group B and the control group was(2.18 ±0.67),(2.60 ±1.14),(0.44 ±0.23),respectively.The content of Sema4 D in vitreous of the two experimental groups was significantly higher than that of the control group.Therefore,there was significant statistical difference(P<0.01).2.The concentration of MMP-9(ng/ml)in vitreous of experimental groups,Group A,Group B and the control group was(3.55±1.71),(2.97±1.64),(0.70±0.29),respectively.The content of Sema4 D in vitreous of the two experimental groups was significantly higher than that of the control group.Therefore,there was significant statistical difference(P<0.01).3.The concentration of VEGF(pg/ml)in vitreous of experimental groups,Group A,Group B and the control group was(237.36±91.20),(920.12±166.51),(144.26±45.59),respectively.The content of Sema4 D in vitreous of the two experimental groups was significantly higher than that of the control group.Therefore,there was significant statistical difference(P<0.01).Summary:1.Sema4 D,MMP-9 and VEGF is highly expressed in the vitreous of proliferative diabetic retinopathy.2.The expression of VEGF in the vitreous cavity can be significantly reduced by the anti-VEGF drug,but there is no statistical significance for the expression level of Sema4 D.Part II The effect and significance of high glucose culture on the expression of Sema4D/Plexin-B1 in retinal pigment epithelium Objective:To establish the RPE cell model in high glucose culture environment to detect the expression of Sema4 D,Plexin-B1,MMP-9 and VEGF in RPE cells under different glucose concentration and culture time.Method:1.Human retinal pigment epithelium(ARPE-19)was selected as the research object,and the cells in logarithmic phase were randomly divided into two groups:normal glucose concentration group(control group)and high glucose concentration group(experimental group).2.Under the condition of 5.56 mmol/L glucose(control group)and 25 mmol/L glucose(high glucose group),the cells were cultured and detected for 12 h,24h and48 h,respectively.3.The content of Sema4 D,VEGF and MMP-9 in supernatant of RPE cell culture fluid was detected by ELISA method.The expression location of Sema4 D,Plexin-B1,MMP-9 and VEGF in RPE cells was observed by immunofluorescence staining.The average gray value of Sema4 D,Plexin-B1,MMP-9 and VEGF protein in RPE cells was detected by Western blot.Result:1.Sema4 D concentration(ng/ml)was detected at 12 h,24 h and 48 h.In the control group,it was(0.45±0.83),(0.43±0.16)and(0.59±0.07),respectively.Further,in the experimental group,it was(0.49±0.03),(2.15±0.24)and(4.19±0.95),respectively.There was significant statistical difference between the groups detected at 12 h and 24h(P<0.01).2.MMP-9 concentration(ng/ml)was detected at 12 h,24 h and 48 h.In the control group,it was(0.28±0.04),(0.35±0.08),(0.31±0.05),respectively.Further,in the experimental group,it was(1.02±0.18),(1.66±0.31)and(1.96±0.21),respectively.There was significant statistical difference between the groups detected at 12 h,24h and 48h(P<0.01).3.VEGF concentration(pg/ml)was detected at 12 h,24 h and 48 h.In the control group,it was(194.85±5.51),(203.83±7.43),(220.99±19.55).Further,in the experimental group,it was(651.78±149.57),(1036.37±168.41)and(1071.40±117.04),respectively.There was significant statistical difference between the groups detected at 12 h,24h and 48h(P<0.01).4.Immunofluorescence staining indicated that Sema4 D and Plexin-B1 in normalRPE cells were mainly located in the cytoplasm and cytomembrane.The VEGF and MMP-9 were located in the cytoplasm,and it was more obvious near nucleus.In the control group,there was a small amount of weak fluorescent staining in all indexes,and the fluorescence staining intensity was significantly enhanced in the experimental group.5.Western blot results implied that at the time of 12 h,24 h and 48 h,the average gray value of Sema4 D in the control group was(0.36±0.02),(0.41±0.02),(0.41±0.09),in the experimental group,it was(0.72±0.02),(0.88±0.05),(0.71±0.18),respectively.There was significant statistical difference between the groups detected at 12 h,24h and 48h(P<0.01).6.Western blot results implied that at the time of 12 h,24 h and 48 h,the average gray value of Plexin-B1 in the control group was(0.32±0.07),(0.44±0.05),(0.47±0.20),in the experimental group,it was(0.69±0.06),(0.90±0.11),(0.89±0.23).There was significant statistical difference between the groups detected at 12 h,24h and 48h(P<0.01).7.Western blot results implied that at the time of 12 h,24 h and 48 h,the average gray value of MMP-9 in the control group was(0.25±0.05),(0.29±0.09),(0.39±0.12),in the experimental group,it was(0.78±0.10),(0.81±0.13),(0.78±0.15),There was significant statistical difference between the groups detected at 12 h,24h and 48h(P<0.01).8.Western blot results implied that at the time of 12 h,24 h and 48 h,the average gray value of VEGF in the control group was(0.33±0.11),(0.42±0.15),(0.60±0.10),in the experimental group,it was(0.80±0.22),(0.84±0.11),(0.90±0.09).There was significant statistical difference between the groups detected at 12 h,24h(P<0.01).Summary:1.Sema4 D,MMP-9 and VEGF was highly expressed in the supernatant of RPE cells cultured with high glucose.2.The high glucose culture state can increase the protein expression of Sema4 D,Plexin-B1,MMP-9 and VEGF in the cells.Part Ⅲ Effect of siRNA silencing Sema4 D on cultured retinal pigment epithelium with high glucose Objective:To target the Sema4 D expression of silencing RPE cells,verify the inhibitory effect,and investigate the effect of Sema4 D,Plexin-B1,MMP-9 and VEGF expression on ARPE-19 cells induced by high glucose environment.Method:1.The human retinal pigment endothelial cell line ARPE-19 was transferred by synthetic siRNA.At the same time,the untreated blank control group and the negative control group(NC group)transferring the insignificant siRNA fragment.Further,the fluorescence quantitative PCR was applied to detect the expression of Sema4 D mRNA,so as to verify the transfection efficiency.Besides,the sequence with the best effect of targeted silencing Sema4 D was selected to establish the successful transferred cell model.2.Under the condition of 5.56 mmol/L glucose(control group)and 25 mmol/L glucose(high glucose group,HB group),the cells were cultured for 48 h.The cells were divided into six groups: control group,control+NC group,control+Sema4D siRNA group,HG group,HG+NC group,HG+Sema4D siRNA group。3.The content of VEGF and MMP-9 in supernatant of RPE cell culture fluid was detected by ELISA method.The expression location of Plexin-B1,MMP-9 and VEGF in RPE cells was observed by immunofluorescence staining.The average gray value of Plexin-B1,MMP-9,VEGF,p-P38 and p-JNK protein in RPE cells was detected by Western blot.Result:1.The expression level of Sema4 D mRNA in siRNA-Sema4 D cell group was lower than in control group and NC group with significant difference(P <0.01).2.The results were in the order of the above six groups,MMP-9 concentration(ng/ml)was(0.33±0.12),(0.28±0.06),(0.36±0.20),(2.01±0.30),(1.89±0.26),(1.12±0.33),respectively.The result of HG+Sema4D siRNA group was lower than HG group and HG+NC group,the difference was statistically significant(P<0.01).3.VEGF concentration(pg/ml)was(265.13±30.52),(204.89±17.66),(303.09±70.64),(1132.31 ± 125.97),(989.47 ± 165.54),(920.56 ± 105.68),respectively.The difference between HG group,HG+NC group and HG+Sema4D siRNA group was no statistical significance(P>0.05).4.Immunofluorescence staining indicated that Plexin-B1 and MMP-9 was significantly enhanced in the experimental group than in the control group.The degree of enhance in HG+Sema4D siRNA group was lower than in HG group and HG+NC group(P<0.01).The fluorescence staining intensity of VEGF was enhanced in the experimental group than in the control group,but the difference between HG group,HG+NC group and HG+Sema4D siRNA group was no statistical significance(P>0.05).5.Western blot results implied that the average gray value of Plexin-B1 in the six groups was(0.44±0.17),(0.53±0.77),(0.39±0.26),(0.85±0.4),(0.81±0.20),(0.45±0.56).There was significant statistical difference between the HG+Sema4D siRNA and HG group(P<0.01).6.Western blot results implied that the average gray value of MMP-9 in the six groups was(0.41±0.03),(0.36±0.02),(0.37±0.02),(0.76±0.04),(0.80±0.15),(0.46±0.06).There was significant statistical difference between the HG+Sema4D siRNA and HG group(P<0.01).7.Western blot results implied that the average gray value of VEGF in the six groups was(0.38±0.06),(0.35±0.03),(0.42±0.02),(0.84±0.04),(0.83±0.04),(0.76±0.17).The average gray scale of VEGF was enhanced in the experimental group than in the control group,but the difference between HG group,HG+NC group and HG+Sema4D siRNA group was no statistical significance(P>0.05).8.The average gray scale of p-P38 in Sema4 D siRNA group was lower than in HG group and HG+NC group,the difference was statistical significance(P<0.05),and there was no significant statistical difference in p-JNK between the three groups.(P>0.05).Summary:1.The expression of Sema4 D in RPE cells can be effectively silenced by siRNA transfection,which can be employed in subsequent experiments.2.The overexpression of Plexin-B1 and MMP-9 in RPE cells with condition of high glucose can be inhibited by knock down Sema4 D.3.The overexpression of VEGF in RPE cells with condition of high glucose were not influenced by knock down Sema4 D.4.The overexpression of MMP-9 in RPE cells can be regulated by Sema4 D through p38 MAPK pathway.Conclusion:1.This study evaluated the expression of Sema4 D in the vitreous of PDR patients and non-PDR patients.We have identified the high expression of Sema4 D in the PDR patients.It may be involved in the development of PDR and retinal neovascularization.2.With the hRPE cell model in high glucose culture environment,the study show the high expression of Sema4 D,Plexin-B1,VEGF and MMP-9.3.The hRPE cell was transferred by synthetic siRNA,Silencing Sema4 D gene can decrease Plexin-B1 and MMP-9 gene expressions through p38 signaling pathway.The silencing effect on gene expression of Sema4 D can provide a new method for the study on DR. |
