| Part I GEO database analysis of HGF content and function prediction in ocular tissues of AMD patients and normal subjectsObjective:To compare the expression differences of HGF and VEGF in AMD patients and normal human eyes through the GEO database,and to analyze the interaction between the AMD patients with enhanced expression in the KEGG database,and to obtain signal pathways that may be related to the pathogenesis of AMD.Methods:In the GEO database,the "Age-related macular degeneration" was used as the search term to screen the chip samples and species.According to the sample size and matching degree,the chip data GSE29801 with the largest sample size was obtained,and the chip was used to visualize the data.The expression of HGF and VEGF was compared between the groups.The KEGG database was used to analyze the expression-enhancing gene set in AMD patients,and the signal pathways related to AMD were obtained by comparing with the known signal pathways in the organism.Results:(1)Analysis of the expression of HGF in the microarray showed that the expression levels of AMD and normal human eyes in the retinal tissue of the macular area were 14.75±7.29 and 12.53±7.30,which were 8.97±2.66 and 9.06±3.32 expressed in the RPE-choroid complex outside the macular area.The expression levels in the RPE-choroid complex of the macular area were 10.96±6.70 and 9.86±3.45(P>0.05).There was a significant difference in the expression of HGF in the retina of the macula(16.96±9.05 vs 11.78±5.10,P<0.05).The expression of HGF in the macular retina of AMD patients was significantly increased.(2)The expression analysis of HGF showed that the expression levels of AMD patients and normal human eyes in the retinal tissue outside the macular area were 183.38± 115.68,140.366±86.9,respectively,the expression levels in the RPE-choroid complex outside the macular area were 135.03.The expression levels of ±55.36 and 105.06±49.94 in the retinal tissue of the macular area were 160.11 ±104.72 and 111.53±57.91.The expression levels in the RPE-choroid complex of the macular area were 125.96±41.58 and 112.47±42.44,respectively.The overall expression of VEGF in AMD patients showed an increasing trend,and the difference was statistically significant in the RPE-choroid complex and macular retinal tissue outside the macular area(P<0.05).(3)KEGG results:Compared with the retinal tissue in the macular area of normal eyes,there were 1058 gene clusters in the macular retinal tissue of AMD patients,and 534 gene sets were up-regulated,and the expression of 12 gene sets was significantly increased(P<0.01)and 49 gene sets was enhanced(P<0.05).It was found that the expression of S1P signaling pathway,Sema4D-related signaling pathway and Wnt signaling pathway was significantly enhanced in the macular retinal tissue of AMD patients.Conclusion:GEO database analysis showed that the expression of HGF in the macular retinal tissue of AMD patients was enhanced,and the expression of VEGF in RPE-choroid complex and retinal tissue was enhanced,indicating that HGF and VEGF are involved in the occurrence of AMD.KEGG Database analysis found that some genes in AMD patients with enhanced gene chip expression are involved in the biological processes of endothelial cell proliferation and migration and angiogenesis.Part Ⅱ Changes of HGF content in human retinal pigment epithelial cells under hypoxic conditions and effects of exogenous HGF protein on RF/6A cellsObjective:To investigate the expression of HGF and VEGF and the secretion of cytokines in ARPE-19 cells at different time points of hypoxia,and to study the effects of exogenous HGF on the proliferation,migration and tubular formation of RF/6A cells.Methods:(1)The 7th to 8th generation ARPE-19 cells with good long-term status were divided into normal control group and anoxic treatment group.300μM cobalt chloride was added for hypoxia treatment,and collected for 0h,2h,4h,8h,12h.mRNA and protein of ARPE-19 cells at various time points,cell supernatants were collected after 24h.Real-time RCR and Western Blot were used to detect the mRNA and protein expression of HGF and VEGF in cells.The protein content of HGF,VEGF,CCL11 and CCL24 in cell supernatant was detected by ELISA.(2)The 7th to 8th generation RP/6A cells with good long-term status were divided into normal control group,5 ng/ml HGF protein treatment group,25 ng/ml HGF protein treatment group and 50 ng/ml HGF protein treatment group.In 96-well plates and Transwell chambers,cultured in different exogenous HGF protein concentrations,MTT assay was used to detect the proliferative capacity of RF/6A cells,Transwell chamber assay was used to detect the migration ability of RF/6A cells,and matrigel gel formation assay was used.RF/6A tubeResults:(1)mRNA expression of ARPE-19 cells after different time of hypoxia:Compared with normal control group,the mRNA level of HGF increased after 2 h(P<0.05).After 4 h and 8 h of hypoxia,mRNA level continued to increase(P<0.05),and the mRNA level reached a peak after 12h of hypoxia(P<0.05).After 12h,the mRNA level gradually decreased(P<0.05).The mRNA level of VEGF only increased at 2h and 4h after hypoxia(P>0.05),and increased significantly from 8h(P<0.05),and continued to 24h(P<0.05).(2)Protein expression of ARPE-19 cells after different time of hypoxia:Compared with normal control group,the protein expression of HGF did not change significantly at 2h and 4h after hypoxia(P>0.05),and gradually increased from 8h after hypoxia,continued until 24 h after hypoxia(P<0.05).The expression of VEGF protein was not significantly changed at 2h,4h and 8h after hypoxia(P>0.05).The expression of VEGF protein was significantly increased after hypoxia for 12h and 24h(P<0.05).(3)The levels of HGF,VEGF,CCL11,CCL24 and other cytokines in ARPE-19 cells were significantly increased after 24 h of hypoxia(P<0.05);(4)5 ng/ml exogenous HGF protein to RP/6A cells had no significant effect on proliferation,migration and angiogenesis(P>0.05).From 25 ng/ml concentration,HGF protein gradually showed proliferative,migration-promoting and tubular formation ability,50 ng/ml HGF protein.The ability to promote tubular formation was stronger than 25 ng/ml(P<0.05).Conclusion:The expression of HGF and VEGF are significantly increased under hypoxic conditions,and the secretion of cytokines HGF,VEGF,CCL11 and CCL24 are increased,indicating that the above cytokines are involved in the hypoxia injury process of RPE cells;HGF protein can pass Regulating the function of endothelial cells and participating in the formation of CNV,several of the above cytokines may affect the occurrence of AMD.Part III The effect of HGF siRNA transfection on human retinal pigment epithelial cells on RF/6A cells under co-culture conditions and its related mechanismsObjective:To investigate the effects of HGF siRNA transfection on ARPE-19 cells on the proliferation,migration and tubular formation of RF/6A cells under co-culture system,and to observe the changes of D114/Notch signaling pathway.Methods:(1)The 7th to 8th generation ARPE-19 cells with good growth status were divided into normal control group,experimental group,negative control group and transfection reagent control group.Real-time RCR and Western Blot were used to detect HGF siRNA against mRNA.And protein silencing efficiency;(2)In co-culture system,7th to 8th generation ARPE-19 cells with good growth status were divided into normal control group,cobalt chloride anoxic treatment group and cobalt chloride+ HGF siRNA treatment group.To observe the effects of ARPE-19 cells on the proliferation,migration and tubular formation of RF/6A cells,and detect the changes of HGF,VEGF,CCLs and D114/Notch signaling pathways in ARPE-19 cells.Results:(1)Real-time PCR and Western Blot results showed that compared with the normal control group,the expression of HGF in the RPE cells of the experimental group was significantly inhibited(P<0.05),and the mRNA and protein levels were decreased by 82%and 66%,respectively.The expression of HGF gene and protein in RPE cells of negative control group and transfection reagent control group did not change significantly(P>0.05),and transfection reagent and siRNA had no obvious side effects on cell growth;(2)In the co-culture system,the proliferation,migration and tubular formation ability of RF/6A cells in the cobalt chloride anoxic treatment group were enhanced(P<0.05).The cobalt/HGF siRNA treatment group RF/6A proliferation,migration and tubular type decreased significantly(P<0.05);(3)After 24 hours of co-culture,the mRNA expressions of HGF,VEGF,CCL11 and CCL24 in ARPE-19 cells were significantly increased under hypoxia(P<0.05),and the mRNA expression of D114 and Notch1 in D114/Notch signaling pathway was observed.There was a rise(P<0.05),but there was no significant change in the expression of Notch4(P>0.05).After HGF siRNA transfection,the expression of HGF and VEGF mRNA in ARPE-19 cells were significantly decreased(P<0.05),the expression of CCL11 and CCL24 was decreased(P>0.05),and the D114/Notch signaling pathway was down-regulated(P<0.05).Conclusion:HGF siRNA can significantly inhibit the expression of HGF gene and protein in ARPE-19 cells,and can reverse the enhancement of hypoxia-induced RF/6A cell function.This HGF may regulate D114/Notch signaling pathway and VEGF expression.The function of endothelial cells. |
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