| BackgroundAge-related macular degeneration is the main cause of visual impairment among the elderly over 60 years of age,classified into two types,one is dry(atrophic),accounting for 85%-90%of AMD,called as geographic atrophy,formed by the lack of retinal pigment epithelium cells and photoreceptor cells.The other one is neovascular(wet),formed by choroidal neovascularization,which leads to hemorrhage and leakage to proliferate between RPE and the retinal.According to the World Health Organization,AMD has become the third main cause of blindness worldwide,following the cataract and glaucoma.In recent years,the morbidity of AMD patients in China has been increasing year by year.With the aging of our population,the prevalence of AMD will be further increased;the majority of affected patients have no effective treatment.Anti-vascular endothelial growth factor(VEGF)treatment is currently the most effective way to inhibit neovascularization and control of vascular leakage,but only 40%of patients can improve vision.In addition,the therapeutic response of CNV patients to anti-VEGF treatment shows great difference,and there is no exact and effective treatment for GA.These are the huge burdens on patients and families of the physical,psychological,economic,quality of life,social interaction,and so on.At present,genetic factors,oxidative stress injury factors,immune factors are considered to be the leading cause of AMD,but its exact pathogenesis is not entirely clear.It is unknown whether abnormalities in the expression of biological molecules,caused by abnormalities in the expression of some genes in the eyes and in the body,can forewarn the occurrence and course of AMD.Identifying the disease-associated genes,traditional study methods often focus on one or some of the genes of interest,resulting in low effective study.With the emergence of the omics method,the springing up of massive data provides us with an important study platform,which greatly improves the efficiency of identifying the disease-associated genes.By means of computational biology,we are able to discover important information from these massive data,helping us clarify the molecular mechanisms and pathogenesis of the disease.Gene expression profile provides us with an unprecedented opportunity to investigate gene expression regulation,such as changes in gene expression during disease development and progression.To further investigate the molecular pathogenesis of AMD and identify AMD-associated biomarkers,and then to provide a scientific basis for the diagnosis,treatment and prevention of AMD,this study based on bioinformatics method,mined and analyzed the transcriptome data of AMD patients,then identified the genes that may play an important role in the pathogenesis of AMD before validating them,performed functional analysis of associated genes.It also discussed the mechanism of differentially expressed genes in the development and progression of AMD,further discussed the efficacy of different clinical treatment programs for AMD,and finally provided guidance for clinical diagnosis and treatment.In the first chapter,we performed a meta-analysis by means of the published gene expression profile microarray data of the NCBI high-throughput gene expression omnibus(Gene Expression Omnibus,GEO).The raw microarray data were from the GSE76237 and GSE29801 expression profile microarray data,which were collected in GEO.The Agilent-014850 Whole Human Genome Microarray 4x44K G4112F expression profile was used for detection.In this study,data were integrated and analyzed from the two aspects of whole blood monocyte expression profiles and lesion tissue expression profiles respectively.Then,we identified the differentially expressed genes(DEGs)and performed a functional analysis of them.Based on the first chapter,in the second chapter we selected 8 candidate genes from the common differentially expressed genes(DEGs),which were obtained through meta-analysis of gene expression profile data of the AMD whole blood mononuclear cell samples and lesion tissue samples from GEO.To validate the expression of these candidate genes in Chinese AMD patients:Real time polymerase chain reaction(Real-time PCR or qPCR)was used to validate the DEGs in the peripheral blood mononuclear cells(PBMC)of the experimental group of Chinese AMD patients and the control group of Chinese healthy human.And then determined whether there was significant difference in the expression of differentially expressed genes in PBMCs of Chinese AMD patients or not.The experimental group was further divided into different groups according to age,sex and therapeutic effect,which were compared and analyzed respectively.In the third chapter,patients with clinical criteria for inclusion in the standard were treated with anti-VEGF drug intravitreal injection and photodynamic therapy(PDT)combined with anti-VEGF intravitreal injection,respectively.Before and after treatment,the best corrected visual acuity(BCVA),the rate of improvement in visual acuity in the operating eye,ocular hemodynamic changes,central fovea thickness(CFT)and visual field parameters were detected in 1 week and 6 months respectively.The difference of curative effect between the two treatment regimens was compared in order to find a more secure and effective AMD treatment program,and to provide a theoretical basis for developing a new AMD treatment program.Part?Analysis of the differences in Gene Expression Profiles between AMD Patients and Healthy HumanObjectiveThrough the analysis of the differences in gene expression profiles of AMD patients and healthy human,we identified the genes that might be associated with the pathogenesis of AMD and performed functional analysis.MethodsBased on the published gene expression profiles microarray data in NCBI GEO,the raw microarray data was from the GSE76237 and GSE29801 expression profile microarray data collected in GEO.GSE76237 contained the monocytes from 14 cases of AMD patients and 15 cases of normal human,using Affymetrix Gene1.0 ST microarray for expression profile analysis;GSE29801 contained retinal-choroidal tissue samples donated by 177 cases of AMD patients and 118 cases without eye disease.Agilent-014850 Whole Human Genome Microarray 4x44K G4112F expression profile microarray was used for detection.Integrated and analyzed the expression profile microarray data separately from the two aspects of whole blood mononuclear cells and lesion tissue samples.The data analysis methods include:1.Microarray data normalized analysis:Bioconductor limma package;2.Differential gene analysis:Bioconductor limma package;3.GO and KEGG functional enrichment analysis:WEB-based GEne SeT Ana Lysis Toolkit;4,Mapping:R language,derived differentially expressed genes(DEGs),and analyzed the function of them.Results1.A total of 111 differentially expressed genes were selected by bioinformatics analysis of whole blood mononuclear cell expression profiles of AMD patients and normal human with fold change>1.5 and corrected P<0.05.Among them,28 were up-regulated and 83 down-regulated;8 were differentially expressed genes with fold change>2,among which 2 were up-regulated and 6 down-regulated.2.The results of GO functional enrichment analysis of the differentially expressed genes of peripheral blood mononuclear cells of the AMD patients showed that the main biological functions of differentially expressed genes involved included leukocyte activation,lymphocyte activation,T cell activation and T cell recruitment.3.The results of KEGG enrichment analysis showed that the differentially expressed genes were mainly involved in hematopoietic cells,T cell receptor pathway,Th1 and Th2 cell differentiation,Th17 cell differentiation and primary immune deficiency signaling pathways.4.A total of 12 differentially expressed genes were selected by bioinformatics analysis of the expression profile microarray of retina-choroidal tissue samples of AMD donors and healthy donors with corrected P<0.05 and fold change>2,among which 3 were up-regulated and 9 down-regulated.5.Analyzed the significantly differential genes filtered from the mononuclear cells of AMD patients with fold change>2 and corrected p<0.05 and those from tissue samples with fold change>2 and corrected P<0.05.Drew Veen diagram.Filtered the common differentially expressed genes of the both groups and removed the probe information with annotation.Then we gained no common differentially expressed genes.Conclusions1.The major biological functions in which the differentially expressed genes of peripheral blood mononuclear cells in AMD patients are involved include leukocyte activation,lymphocyte activation,T cell activation,and T cell recruitment.The major signaling pathways include hematopoietic cells,T cell receptor pathway,Th1 and Th2cell differentiation,Th17 cell differentiation and primary immunodeficiency.These suggest that differentially expressed genes may be closely associated with the immune response of AMD patients.2.The gene expression has different characteristics between mononuclear cells themselves and diseased tissue,but these differential genes are not repeated.This may be associated with the onset of AMD to stimulate the systemic immune system,involved in mononuclear cells and diseased tissue,but there were different gene expression.Part t?Study on the Transcriptome of AMD PatientsObjectiveThrough the study on the transcriptome of AMD patients and healthy human,we investigated the influencing mechanism of differentially expressed genes on the pathogenesis and prognosis of AMD to provide experimental basis for finding new diagnostic and therapeutic targets,and laid experimental foundation for the study of AMD diagnostic index and prognosis index.MethodsAccording to the results of the first chapter,we first selected 8 genes)from the differentially expressed genes(DEGs),which were gained through the meta-analysis of the gene expression profile data of AMD whole blood mononuclear cells samples from GEO by bioinformatics methods.Then by real time polymerase chain reaction(Real-time PCR or qPCR),we validated these DEGs in PBMCs of the experimental group samples with 21 cases of Chinese AMD patients and the control group samples with 15 cases of healthy human.Then we determined whether there was a difference in the expression of differentially expressed genes in PBMCs of Chinese AMD patients or not.The experimental group was further divided into different groups according to age,sex and treatment effect.The experimental data were validated and compared respectively.Results1.Among the eight candidate differentially expressed genes,the results of three genes in the validated samples were consistent with the data mining results.The expression of TMEM176A,TMEM176B and ZEB2 was significantly up-regulated in PBMCs of AMD(P<0.001).The results of the other five candidate DEGs in the validated samples were not consistent with the data mining results.The expression of LEF1,FCMR,MS4A1,CD3G and IL7R was significantly up-regulated in PBMCs of AMD patients(P<0.05).2.The AMD patients were divided into different groups by sex,which were compared and analyzed.The results showed that,the expression levels of 8 candidate genes in AMD and PBMC patients of different sex groups were significantly different from those of healthy people,and the differences were statistically significant(P<0.01).TMEM176A?FCMR?CD3G?MS4A1?IL7R?ZEB2 and LEF1 gene expression levels of female patients were higher than those of male patients,but the difference was not statistically significant(P>0.05);The expression levels of TMEM176B in male patients were higher than those in female patients,but the difference was not statistically significant(P>0.05).This indicated that there was significant difference in gene expression in PBMCs of patients with different sexes,but no significant correlation was found between DEGs expression and sex.3.AMD patients were divided into different groups by age,which were compared and analyzed.The results showed that the expression levels of 8 candidate genes in AMD and PBMC patients of different age groups were significantly different from those of healthy people and the differences were statistically significant(P<0.05).The expression levels of TMEM176A and TMEM176B were higher in patients with age>70 years than those with age?70 years,but there was no significant difference between the two groups(P>0.05).The gene expression levels of MS4A1 were significantly higher in patients with age?70 years than those with age>70 years.The difference was statistically significant(P<0.05).The expression levels of LEF1?FCMR?CD3G?IL7R and ZEB2 in PBMCs were higher in patients with age?70 years than those with age>70 years,but there was no significant difference between the two groups(P>0.05).This indicates that there was significant difference in gene expression in PBMCs of patients with different ages.The MS4A1gene expression levels were significantly associated with the age of the patient.This result suggests that the occurrence of AMD was associated with aging.4.AMD patients were divided into two different groups according to the efficacy,which were compared and analyzed.The results showed that the expression levels of the eight genes were higher in patients with poor efficacy than those with good curative effect,but the difference was not statistically significant(P>0.05).This indicates that the eigtht DEGs expression levels were associated with the curative effect of patients,but not significant.This suggests that we may filter genes from them as the assessment indicators of AMD prognosis,but we need do more studys.Conclusions1.In this study,eight genes(TMEM176A,TMEM176B,ZEB2,LEF1,FCMR,MS4A1,CD3G and IL7R)expressions are significantly up-regulated in PBMCs of AMD.The function of these differentially expressed genes mainly related to membrane receptors and related signal transduction,its encoding protein is mainly distributed in the outer cell membrane,differentiation of biological processes involved mainly involved in immune response and immune cells.All these suggest that the occurrence and development of AMD are closely related to systemic immune response.2.There was significant difference in DRGs expression in PBMCs of patients with different sexes,but no significant correlation was found between DEGs expression and sex.There was no significant gender difference in the progression of AMD.3.There was significant difference in DRGs expression in PBMCs of patients with different ages.The MS4A1 gene expression levels were significantly associated with the age of the patient.This result suggests that the occurrence of AMD was associated with aging.4.The eigtht DEGs expression levels were associated with the curative effect of patients,but not significant.This suggests that we may filter genes from them as the assessment indicators of AMD prognosis,but we need do more studys.Part?Study on the efficacy of ant-VEGF drugs intravitreal injection combined with PDT in the treatment of AMDObjectiveTo find a more secure and effective AMD treatment program to provide a theoretical basis for the development of new AMD treatment program.MethodsAccording to the inclusion criteria and exclusion criteria,96 patients(192 eyes)who were diagnosed with AMD at the First Affiliated Hospital of Zhengzhou University from January 2013 to January 2016 were selected.According to the treatment plan,the patients were randomly divided into observation group and control group,each group of 48 patients(96 eyes).The patients in the control group were treated with anti-VEGF drugs and received intravitreal injection of 0.5 mg/0.05 ml leuzorab.According to the 3+prn regimen,once a month for 3 consecutive injections,and then repeated the injection if there needed.The patients in the observation group were treated with anti-VEGF drugs combined with PDT.PDT treatment was performed according to the method of photodynamic therapy for AMD group(TAP).The dosage of vildenafil was 6 mg/m2 calculated by TAP method,the shock wavelength was 689nm,irradiance 600mw/cm~2,energy 50J/cm~2,irradiation time 83S.Intravitreal injection of 0.5 mg/0.05ml dexamethasone was performed within 3days after PDT treatment.The best corrected visual acuity,the rate of improvement in visual acuity in the operating eye,ocular hemodynamic changes,central foveal thickness(CFT)and visual field parameters were measured before and after 1 week to 6 months after treatment.SPSS 23.0 statistical software was used to analyze the data of this study.Results1.After treatment,the best-corrected visual acuity of both groups of patients was slowly increased.Within 1 to 6 months after surgery,the best-corrected visual acuity was significantly higher in the observation group than that in the control group.The difference was statistically significant(P<0.05).The proportion of visual acuity increased in the observation group was higher than that in the control group.The difference was statistically significant(P<0.05).2.Color Doppler ultrasonography showed that the peak systolic velocity(PSV)and the end diastolic velocity(EDV)of the posterior ciliary short arteries(PCA)in the observation group were higher than those in the control group(P<0.05).The arterial resistance index(RI)and pulsatility index(PI)of the observation group were lower than those of the control group.The difference was statistically significant(P<0.05).3.The central foveal thickness(CFT)in the observation group was lower than that in the control group within 6 months after treatment(P<0.05).With visual parameters as 10°and 4°,the mean sensitivity(MS)in the observation group was higher than that in the control group.The difference was statistically significant(P<0.05).The absolute value of the mean defect of visual field(MD)in the observation group was lower than that in the control group.The difference was statistically significant(P<0.05).Conclusions1.Anti-VEGF drugs combined with PDT therapy can significantly improve the best-corrected visual acuity.It has an obviously curative effect on AMD.2.Anti-VEGF drugs combined with PDT therapy can improve the PCA hemodynamic parameters;furthermore the improved blood supply can reduce the macular edema,reduce the central foveal thickness(CFT)value,and further reduce visual field defects.Therefore it has a high clinical value.3.On the premise of inhibiting neovascularization and protecting vision,anti-VEGF drugs combined with PDT therapy has reduced vitreous cavity injection and decreased the incidence of eye complications.However,whether the combined treatment regimen is safer for AMD is still not conclusive and needs further study. |
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