【作者】 刘骁；

【导师】 唐罗生；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 目的：研究湖南地区2型糖尿病视网膜病变(diatetic retinopathy,DR)遗传及环境易感因素；筛选糖尿病视网膜病变发生的高危人群。(1)采用荟萃分析方法,搜寻黄种人中与糖尿病视网膜病变发生相关的血管内皮生长因子(vascular endothelial growthfactor,VEGF)单核苷酸多态性位点。(2)探讨湖南地区人群中VEGF单核苷酸多态性位点与糖尿病视网膜病变易感性的关联性。(3)探讨血清中VEGF蛋白总量,亚型VEGF165及其异构体VEGF165b含量与糖尿病视网膜病变的相关性。(4)采用病例对照及logistic回归分析比较VEGF单核苷酸多态性位点、VEGF蛋白及其他人口学特征和生化指标,探讨糖尿病视网膜病变发生的高危因素。方法：(1)回顾2012年3月前有关VEGF单核苷酸多态性与糖尿病视网膜病变的所有英文文献。系统性检索及获取原始文献、制定纳入及排除标准；将单核苷酸多态性位点与糖尿病视网膜病变相关的实验数据进行荟萃分析。(2)根据荟萃分析显示黄种人中有意义的VEGF单核苷酸多态性位点,采用病例对照方法,使用限制性片段长度多态性聚合酶链反应(PCR-RFLR)检测其在湖南地区正常人、糖尿病无视网膜病变患者(diabetic without retinopathy, DWR)、糖尿病视网膜病变患者(diabetic retinopathy, DR)的分布,通过卡方检验统计分析其在各组间的分布差异。(3)采用病例对照方式,使用酶联免疫吸附测定法检测湖南地区正常人,糖尿病无视网膜病变患者,糖尿病视网膜病变患者的血清中VEGF总量,亚型/EGF165及其异构体VEGF165b含量,通过成组T检验及线性回归分析统计分析其含量在各组间的分布差异,及与、/EGF单核苷酸多态性位点及环境因素间的关联性。(4)采用病例对照方式,统计糖尿病无视网膜病变患者,糖尿病视网膜病变患者的人口学特征及生化指标的相关数据及VEGF单核苷酸多态性位点及VEGF蛋白表达,进行成组T检验及logistic回归分析,探讨糖尿病视网膜病发生的高危易感因素。结果：(1)荟萃分析结果：VEGF rs699947共显性模型-杂合型／野生型(CAVs CC)与DR发病风险存在相关性(I2=64%, OR=1.2795%CI [1.05,1.54], P=0.02),尤其在黄色人种中(I2=38%, OR=1.4695%CI [1.17,1.82], P=0.0007)。其它模型与DR发病无显著相关性(P>0.05)VEGF rs1570360各模型在各色人种中与DR的发病无显著相关性(P>0.05)。VEGF rs2010963亚组分析：黄色人种组隐性模型(CC V s GC+GG)显示与DR的发病相关(I2=60%, OR=1.2895%CI [1.01,1.63], P=0.04); PCR-RFLR测序方式组隐性模型(CC V s GC+GG)与DR的发生明显相关(I2=46%, OR=1.5095%CI [1.05,2.14], P=0.03)VEGF rs3025039共显性模型-纯和型／野生型(TT Vs CC)与DR发病风险存在相关性(I2=50%, OR=2.4795%CI [1.11,5.51], P=0.03),特别是在黄色人种中(I2=51%, OR=3.5395%CI [1.35,9.26], P=0.01)。隐性模型(TT Vs CT+CC)白色人种与DR发病无明显相关性(P=0.05),在黄色人种中示与DR的发生相关(I2=33%, OR=3.1995%CI [1.21,8.42], P=0.01);其它各模型与DR的发病无明显相关性(P>0.05)。(2)本省人群中DWR组与DR组VEGF基因多态性位点基因型频率及等位基因频率：VEGF基因rs699947位点：DR组AA基因型分布频率为14.8%,DWR组为4.3%；CC基因型DR组为48.1%,DWR组为60.9%(P=0.04,<0.05),其等位基因A频率在DR组为33.3%,DWR组为21.7%(P=0.016,<0.05)。携带rs699947AA基因型的糖尿病患者发生DR的危险性较rs699947CC基因型及CA基因型携带者高3.83倍(OR=3.83)。VEGF基因rs833061位点：DR组CC基因型分布频率为16.0%,DWR组为4.3%；TT基因型DR组为45.7%,DWR组为58.7%(P=0.025,<0.05),其等位基因C频率在DR组为35.2%,DWR组为22.8%(P=0.011,<0.05)。携带rs833061CC基因型的糖尿病患者发生DR的危险性较rs833061TT基因型及TC基因型携带者高4.21倍(OR=4.21)。VEGF基因rs13207351位点：基因型频率及等位基因频率在研究组和对照组间的差异无明显统计学意义(P>0.05)。携带rs13207351GG基因型、GA基因型及AA基因型的糖尿病患者发生DR的危险性没有明显差别。VEGF基因rs2010963位点：DR组CC基因型分布频率为21.0%,DWR组为9.8%。GG基因型DR组为28.4%,DWR组为42.4%(P=0.049,<0.05),其等位基因C频率在DR组为46.3%,DWR组为33.4%(P=0.017,<0.05)。携带rs2010963CC基因型的糖尿病患者发生DR的危险性较rs2010963GG基因型及GC基因型携带者高2.45倍(OR=2.45)。VEGF基因rs3025039位点：基因型频率及等位基因频率在研究组和对照组间的差异无明显统计学意义(P>0.05)。携带rs3025039CC基因型、CT基因型及TT基因型的糖尿病患者发生DR的概率没有明显统计差异。(3) VEGF蛋白总量、VEGF165含量和VEGF165b含量：1)正常对照组中血清VEGF总量为44.47±3.42(pg/ml), DWR组血清VEGF总量为57.74±11.23(pg/ml), DR组59.25±11.62(pg/ml),DWR组及DR组的VEGF总量都较正常对照组升高(P<0.05),但DWR组与DR组之间VEGF总量没有明显的统计学差异(P>0.05)。2)正常对照组中VEGF165含量为15.89±1.31(pg/ml), DWR组为22.68±4.52(pg/ml); DR组为27.43±7.29(pg/ml),与正常对照组比具有统计学意义(P<0.05)；且VEGF165在D WR组及DR组之间也有显著差异(P=0.025)。3)VEGF165b在血中的含量在正常对照组为9.25±0.82(pg/ml); DWR组为6.97±1.46(pg/ml); DR组为5.43±2.35(pg/ml).其含量在三组之间呈现逐渐降低的趋势,差异具有统计学意义(P<0.05)。VEGF165及VEGF165b的比值在三组间出现变化,比值逐渐增大(1.72、3.25、5.05)。4)蛋白总量与基因位点：携带rs2010963CC基因型人群其血中VEGF蛋白含量较GG基因型及GA型显著增高(CC基因型VEGF蛋白含量为62.59±13.71(pg/ml),GA基因型蛋白含量为53.27±10.37(pg/ml),GG基因型蛋白含量为50.85±10.59(pg/ml));CC基因型与GG基因型及GC基因型比较均具有统计学上的显著性差异,P<0.05),其他各SNP位点各基因型之间无明显统计学差别(P>0.05)。(4)糖尿病视网膜病变易感因素分析：1)DWR组及DR组之间的人口学特征及生化指标的相关数据进行成组T检验,发现收缩压、舒张压、糖化血红蛋白及肌酐值在两组之间存在显著的统计学差异(P<0.05)。2)logistic回归分析：收缩压高于160mmHg的糖尿病患者发生DR的危险性较低于130mmHg的高7.5倍,舒张压高于90mmHg的糖尿病患者发生DR的危险性较低于90mmHg的高2倍。HbAIC含量高于14.0%的糖尿病患者发生DR的危险性较低于8.0%的高11.50倍。肌酐值高于100mmol/L的糖尿病患者发生DR的危险性较低于60mmol/L的高出5.5倍。携带rs2010963CC基因型糖尿病患者发生DR的危险性较GG基因型患者高3.2倍。VEGF165含量高于28.0mmol/L的糖尿病患者发生DR的危险性较低于28.0mmol/L的患者高9.3倍,而VEGF165b含量高于5.0mmol/L的糖尿病患者发生DR的危险性较低于5.0mmol/L的患者明显降低(OR=0.013),VEGF165b对于糖尿病患者有明显保护作用。结论：(1)黄色人种中VEGF rs699947的共显性模型(C A Vs C C)、 VEGF rs2010963隐性模型(CC Vs GC+GG)及VEGF rs3025039的共显性模型(TT Vs CC)、隐性模型(TT Vs CT+CC)跟糖尿病视网膜病变的发生相关；而在白色人种中上述多态性位点与糖尿病视网膜病变无明显相关性。(2)本省人群VEGF基因rs699947位点、VEGF基因rs833061位点、VEGF基因rs2010963位点的多态性变化在糖尿病视网膜病变发生发展中具有一定的意义(3)VEGFXXX与EGFxxxb比值改变导致糖尿病视网膜病变的发生和发展。rs2010963位点多态性变化为功能性改变,对糖尿病患者发生糖尿病视网膜病变有重要意义。(4)收缩压≥160mmHg舒张压≥90mmHg.糖化血红蛋白≥8.0mmol/L、肌酐值≥VEGF165>28mmol/L、rs2010963CC基因型及VEGF165b<5.0mmol/L为糖尿病视网膜病变发生的高危人群。更多还原

【Abstract】 Objective:The aim of this study is to discuss genetic and environmental predisposing factors of diabetic retinopathy (DR.) with type2diabetes in Hunan Province and screen the high-risk groups of diabetic retinopathy.(1) To Search VEGF single nucleotide polymorphisms related to diabetic retinopathy in the yellow people by meta-analysis method.(2) To investigate the susceptibility between VEGF single nucleotide polymorphisms and diabetic retinopathy in Hunan Province.(3) To investigate correlation between serum VEGF total protein, isoform VEGF165and VEGF165b with diabetic retinopathy(4)To explore the risk factors of diabetic retinopathy through comparing VEGF single nucleotide polymorphisms, VEGF protein and other demographic characteristics and biochemical parameters by case-control and logistic regression analysis.Methods:(1) Review relevant articles about the VEGF single nucleotide polymorphisms and diabetic retinopathy was carried out until March2012. A systematic search of electronic databases and access to the original literature, development of inclusion and exclusion criteria; and making a meta-analysis by the experimental data.(2) According to the result of meta-analysis, detect VEGF single nucleotide polymorphisms among normal people, diabetic patients with no retinopathy and diabetic retinopathy patients by case-control method in Hunan Province,and do statistical analysis of distribution among the groups by the chi-square test.(3) Enzyme-linked immunosorbent assay was used to detect serum total VEGF、VEGF165and VEGF165b among normal people, diabetic patients with no retinopathy and diabetic retinopathy in Hunan province. Do statistical analysis of distribution among them through group T-test and linear regression analysis and discuss correlation with VEGF single nucleotide polymorphisms and environmental factors.(4) Investigate the susceptibility factors of diabetic retinopathy through a case-control based study among diabetic patients with no retinopathy and diabetic retinopathy on risk factor as demographic characteristics、biochemical parameters、VEGF single nucleotide polymorphisms and VEGF protein expression; Statistical analysis of data by group T-test and logistic regression analysis.Results:(1) Meta-analysis:VEGF rs699947A significant association between it and DR risk was found in dominant model (CA Vs CC)(I2=64%,OR=1.27,95%CI[1.05,1.54],P=0.02),especially in the yellow race(I2=38%,OR=1.46,95%CI[1.17,1.82]p=0.0007).Other models with DR was no significant correlation.(P>0.05).VEGF rs1570360All type of models with DR was no significant correlation.(P>0.05).VEGF rs2010963A significant association between it and DR risk was found in recessive model(CC Vs GC+GG)in the yellow race group by subgroup analysis(I2=60%,OR=1.2895%CI[1.01,1.63],P=0.04)；and in recessive model(CC Vs GC+GG)by PCR-RFLR subgroup analysis(I2=46%,OR=1.5095%CI[1.05,2.14],P=0.03)VEGF rs3025039A significant association between it and DR risk was found in co-dominant model (TT Vs CC)(I2=50%,OR=2.47,95%CI[1.11,5.51],P=0.03),especially in the yellow race(I2=51%, OR=3.5395%CI[1.35,9.26],P=0.01).Recessive model(TT Vs CT+CC)with DR was no significant correlation in the white race (P=0.05),but different in the yellow race(I2=33%,OR=3.1995%CI [1.21,8.42],P=0.01).Other models with DR was no significant correlation.(P>0.05).(2)Frequencies of VEGF gene polymorphism genotype and allele between the DWR group and DR group in Hunan province: VEGF rs699947:Frequencies of DR group AA genotype is14.8%, DWR group is4.3%; The CC genotype is48.1%in DR group, DWR group is60.9%(P=0.04,<0.05).The allele A frequency in the DR group is33.3%, and DWR group is21.7%(P=0.016,<0.05). The diabetes patients with rs699947AA genotype occurred the DR risk was3.83times higher than who with CC genotype and CA genotype (OR=3.83).VEGF rs833061:Frequencies of DR group CC genotype is16.0%, DWR group is4.3%; The TT genotype is45.7%in DR group, DWR group is58.7%(P=0.025,<0.05).The allele C frequency in the DR group is35.2%, and DWR group is22.8%(P=0.011,<0.05). The diabetes patients with rs833061CC genotype occurred the DR risk was4.21times higher than who with TTgenotype and TC genotype (OR=4.21).VEGF rs13207351:Frequencies of genotype and allele between the two groups had not statistically significant difference (P>0.05). All rs13207351genotype in patients with diabetes is no significant difference in the risk of DR.VEGF rs2010963:Frequencies of DR group CC genotype is21.0%, DWR group is9.8%; The GG genotype is28.4%in DR group, DWR group is42.4%(P=0.049,<0.05).The allele C frequency in the DR group is46.3%, and DWR group is33.4%(P=0.017,<0.05). The diabetes patients with rs2010963CC genotype occurred the DR risk was2.45times higher than who with GG genotype and GC genotype (OR=2.45).VEGF rs3025039:Frequencies of genotype and allele between the two groups had not statistically significant difference (P>0.05). All rs3025039genotype in patients with diabetes is no significant difference in the risk of DR.(3) VEGF total protein,VEGF165and VEGF165b:1) serum VEGF total protein was44.47±3.42(pg/ml) in normal control group,57.74±11.23(pg/ml) in the DWR group and59.25±11.62(pg/ml) in DR group.Compared with the control group, VEGF total protein in the DWR group and DR group was increased (P<0.05), but the total amount of VEGF between the DWR group and DR group was no statistically significant difference (P>0.05).2) Serum VEGF165was15.89±1.31(pg/ml) in normal control group,22.68±4.52(pg/ml) in the DWR group and27.43±7.29(pg/ml) in DR group.Compared with the control group, VEGF165in the DWR group and DR group was increased (P<0.05).And VEGF165between the DWR group and DR group had statistically significant difference (P=0.025).3) Serum VEGF165b was9.25±0.82(pg/ml) in normal control group,6.97±1.46(pg/ml) in the DWR group and5.43±2.35(pg/ml) in DR group. Content among the three groups showed a gradually decreasing trend, the difference was statistically significant (P<0.05). The ratio of of VEGF165and VEGF165b was changed among the three groups, and the ratio gradually increased (1.72,3.25,5.05).4)The relationship between VEGF protein and VEGF gene: Compared with GG genotype and GA genotype, VEGF protein was significantly higher in the patients with rs2010963CC genotype (62.59±13.71(pg/ml) in the CC genotypes,53.27±10.37(pg/ml) in GA genotype,50.85±10.59(pg/ml) in GG genotype); and the difference had statistically significance (P<0.05).Other SNP genotypes had no statistically significant difference (P>0.05).(4)The susceptibility factors of diabetic retinopathy:1) Systolic blood pressure, diastolic blood pressure, glycosylated hemoglobin and creatinine between the two groups had a statistically significance to analyze demographic characteristics and biochemical parameters between DWR group and DR group by group T-test (P <0.05).2) Logistic regression analysis:diabetic patients with systolic blood pressure over160mmHg had a7.5times higher DR risk than who with130mmHg; diabetic patients with diastolic blood pressure over90mmHg had a2times higher DR risk than who less to90mmHg. Diabetic patients with HbAIC over14.0%had11.50times higher DR risk than with who less to8%. Diabetic patients with Creatinine values above100mmol/L had5.5times higher DR risk than with who less to60mmol/L. Diabetic patients with rs2010963CC genotype had3.2times higher DR risk compared with GG genotype. Diabetic patients with VEGF165above28.0mmol/L had9.3times higher DR risk than with who less to28.0mmol/L. Diabetic patients with VEGF165b above5.0mmol/L had lower DR risk than with who less to5.0mmol/L (OR=0.013).VEGFi65b has a significant protective effect for people with diabetes.5. Conclusions:(1) The co-dominant model (CA Vs CC) of VEGF rs699947, the implicit model (CC Vs GC+GG) of VEGF rs2010963, the co-dominant model (TT Vs CT+CC) and the implicit model (TT Vs CC) of VEGF rs3025039, are associated with the occurrence of diabetic retinopathy in the yellow race, but have no obvious correlation diabetic retinopathy in the white race.(2) The polymorphism variation of rs699947, rs833061and rs2010963of the VEGF gene has significance in the occurrence and development of diabetic retinopathy in the population of Hunan province.(3) The change of the ratio of VEGFxxx and VEGFxxxb leads to the initiation and progression of diabetic retinopathy. The polymorphism variation of rs2010963is a functional change, which is important for occurrence of diabetic retinopathy in diabetic patients.(4) People with SBP≥160mmHg, DBP≥90mmHg, glycosylated hemoglobin≥8.0mmol/L, Creatinine values≥100mmol/L,28mmol/L, rs2010963CC gene and VEGF165b<5.0mmol/L have high risk for diabetic retinopathy.更多还原