糖尿病视网膜病变与脉络膜厚度和PPARγ基因多态性的相关性及其影响因素的研究

发布时间：2018-06-12 11:50

  本文选题：糖尿病视网膜病变 + 脉络膜　； 参考：《天津医科大学》2014年博士论文

【摘要】：目的：应用深度增强成像技术研究不同阶段的糖尿病视网膜病变(diabetic retinopathy, DR)和糖尿病黄斑水肿(diabetic macular edema, DME)黄斑区脉络膜厚度变化,建立视网膜muller细胞原代培养体系,探讨不同阶段DR与PPARy基因单核苷酸多态性的相关性及其影响因素。 方法：选择正常志愿者共120例,年龄25～85岁。根据研究对象不同年龄分组。深度增强成像相干光断层扫描测量脉络膜厚度。分析黄斑中心凹脉络膜厚度(subfoveal choroidal thickness, SFCT)与年龄的相关性。收集2型糖尿病患者92例(92眼),共分为3组：A组(DR-/DME-):无DR无DME组36例(36眼)；B组(NPDR+/DME-):无DME的非增生性糖尿病视网膜病变(Non-proliferative diabetic retinopathy, NPDR)组47例(47眼)；C组(PDR+/DME-):无DME的增生性糖尿病视网膜病变(Proliferative diabetic retinopathy, PDR)组9例(9眼),分别比较组间脉络膜厚度的差异、各组与正常人群脉络膜厚度的差异。选择DME33例33眼,采集指标：空腹血糖、糖化血红蛋白(Glycosylated hemoglobin, HbAlc)、总胆固醇(Total cholesterol, TC)、甘油三酯(Triglycerides, TG)、高密度脂蛋白(High-density lipoprotein, HDL)、低密度脂蛋白(Low-density lipoprotein, LDL)等。比较DME与正常人群及不同阶段DR的脉络膜厚度。分析糖尿病病程、空腹血糖、HbAlc、TG、TC、LDL、HDL、UAER等因素与DME脉络膜厚度变化的相关性。另选择不同阶段DR120例120眼,直接测序法检测PPARγpro12Ala位点基因型,比较不同阶段DR等位基因和基因型频率分布差异,分析等位基因和基因型与DR发生的相关性；分析PPARy基因pro12Ala位点多态性与不同阶段DR的相关性。进一步探讨不同等位基因与TG、LDL、HDL、HbAlc、UAER等临床生化指标的相关性。同时应用改良方法进行体外原代培养并纯化鉴定SD大鼠视网膜muller细胞,为下一步研究提供实验基础。 结果：正常人群平均SFCT为(271.32±35.63)μm男性SFCT平均值为(286.32±35.98)μm,60名女性为(254.33±39.61)μm,男性平均SFCT高于女性,且两者之间差异有统计学意义(P0.05)。不同年龄组之间SFCT差异有统计学意义(P0.001)。随着年龄的增加,SFCT逐渐减小,与年龄呈负相关(r=-0.781,P0.001)。A组：(DR-/DME-)的SFCT为145-306μm,平均为(271.19±34.68)μm,与正常对照人群的平均SFCT进行比较,差异无统计学意义(P0.05)。B组：(NPDR+/DME-)的SFCT为176～336μm,平均为(259.64±37.79)μm,与正常对照的平均SFCT比较差异无统计学意义(P0.05)。C明：(PDR+/DME-)的SFCT为171～289μm,平均(232.98±34.13)μm,与正常对照的平均SFCT比较,脉络膜明显变薄,且差异具有统计学意义(P0.05)。D组：(NPDR+/DME+)的SFCT为156～278μm,平均为(229.64±33.66)μm,与正常对照45～65岁(含)年龄组的平均SFCT(270.78±35.97)μm比较,脉络膜明混变薄,差异具有显著统计学意义(P0.01)。D组与B组及正常人群比较,D组脉络膜明显变薄,且差异具有统计学意义(P0.01)。HbAlc、LDL、UAER水平是DME发生的危险因素,其Exp(B)分别是5.512(P0.001)；2.821(P=0.017)；1.582(P=0.009)。合并DME的NPDR脉络膜厚度与LDL和UAER具有相关性(r=-0.601、-0.673,P0.01),而与糖尿病病程、空腹血糖、HbAlc TG、TC、HDL、肌酐、收缩压及舒张压均无相关性(r-=-0.253、0.219、0.095、0.310、0.185、0.224、0.237、0.421、0.281,P0.05)。通过胰蛋白酶多次反复短时间消化,以谷氨酰胺合成酶为特异性抗体进行细胞鉴定,体外培养的muller细胞形态一致,谷氨酰胺合成酶反应阳性。DR患者PPARy基因G等位基因的分布频率为5.8%,PDR和NPDR患者基因型均以C/C型为主导,所占比例分别为87%和89.2%,二者之间差异无统计学意义。不同基因型全身主要生化指标比较显示：与CC基因型的DR患者相比,G/G或G/C基因型患者UAER水平明显降低,具有统计学差异,而TG、LDL、HDL、HbAlc水平差异无统计学意义。结论：正常中国人平均SFCT为271.32±35.63gm,各年龄段男性平均SFCT高于女性。随着年龄的增加,SFCT逐渐减小,与年龄呈负相关。鼻侧脉络膜均最薄。随着DR加重,脉络膜厚度呈降低趋势。无DR的PDR患者脉络膜较正常人及无DR糖尿病患者均明显变薄,与无DME的NPDR目比,合并DME的NPDR脉络膜显著变薄。LDL和UAER水平与DME脉络膜厚度呈负相关。胰蛋白酶反复消化培养是一种可靠的获得较为纯化、表达稳定的muller细胞体外原代培养的方法。PPARy基因pro12Ala位点的基因型和等位基因的分布频率与DR的发生,以及不同阶段的DR无明确相关性；但PPARγ基因G等位基因可能减低DR患者UAER值,对其肾脏功能可能具有一定保护作用。
[Abstract]:Objective: To study the changes of the choroidal thickness in the macular region of diabetic retinopathy (diabetic retinopathy, DR) and diabetic macular edema (diabetic macular edema, DME) by depth enhanced imaging, and to establish a primary culture system for retinal Muller cells and explore the single nucleotide polymorphisms of DR and PPARy genes at different stages. Correlation and its influencing factors.
Methods: a total of 120 normal volunteers, aged 25~85 years, were divided into different age groups. The depth enhanced imaging coherent light tomography was used to measure choroidal thickness. The correlation between subfoveal choroidal thickness (SFCT) and age was analyzed. 92 cases of type 2 diabetes mellitus (92 eyes) were collected, which were divided into 3. Group A (DR-/DME-): no DR without DME group 36 cases (36 eyes); B group (NPDR+/DME-): non proliferative diabetic retinopathy of DME (Non-proliferative diabetic retinopathy, NPDR) group 47 cases (47 eyes); C group: 9 cases (9 eyes) of proliferative diabetic retinopathy (9 eyes), respectively. The difference of choroidal thickness between the groups and the choroidal thickness of the normal group. Select DME33 33 eyes and collect the indexes: fasting blood glucose, glycated hemoglobin (Glycosylated hemoglobin, HbAlc), total cholesterol (Total cholesterol, TC), triglyceride (Triglycerides, TG), high density lipoprotein (High-density lipoprotein, HDL), Low density lipoprotein (Low-density lipoprotein, LDL) and so on. Compare the choroidal thickness of DME and normal people and different stages of DR. Analysis of the correlation between diabetes course, fasting blood glucose, HbAlc, TG, TC, LDL, HDL, UAER and other factors with the changes of the thickness of the DME choroid. The other 120 eyes were selected at different stages and direct sequencing was used to detect the gamma locus Genotypes, compare the difference of DR allele and genotype frequency distribution at different stages, analyze the correlation between alleles and genotypes and DR, analyze the correlation between pro12Ala polymorphism of PPARy gene and DR at different stages, and further discuss the correlation of different alleles with TG, LDL, HDL, HbAlc, UAER and other clinical biochemical indexes. The Muller cells of SD rat retina were cultured and purified in vitro by improved method, providing experimental basis for further research.
Results: the average SFCT of the normal population was (271.32 + 35.63) mu m male SFCT mean (286.32 + 35.98) mu m, 60 women (254.33 + 39.61) m, and the average male SFCT was higher than the female, and the difference between the two groups was statistically significant (P0.05). The SFCT difference between different age groups was statistically significant (P0.001). As the age increased, SFCT gradually decreased, The negative correlation with age (r=-0.781, P0.001) group.A: (DR-/DME-) SFCT was 145-306 mu m, the average was (271.19 + 34.68) mu m, compared with the average SFCT in the normal control group, the difference was not statistically significant (P0.05).B group: (NPDR+/DME-) SFCT was 176~336 mu, the average was (259.64 + 37.79) micron, and there was no difference compared with the normal control. Statistical significance (P0.05).C clear: (PDR+/DME-) SFCT is 171~289 mu m, average (232.98 + 34.13) mu m, compared with the average SFCT of normal control, the choroid membrane is thinner, and the difference has statistical significance (P0.05).D group: (NPDR+/DME+) SFCT 156~278 mu m, average (229.64 + 33.66) mu, and normal control 45~65 years (containing) age group flat level Compared with SFCT (270.78 + 35.97) mu m, choroidal Ming was thinner and thinner, the difference had significant statistical significance (P0.01), compared with the B group and the normal group, the choroidal membrane in the D group was obviously thinner, and the difference was statistically significant (P0.01).HbAlc, LDL, UAER level was the risk factor of DME, and the Exp (2.821); 1.582 (1.582) 0.009. The NPDR choroid thickness of the combined DME was associated with LDL and UAER (r=-0.601, -0.673, P0.01), but had no correlation with the course of diabetes, fasting blood glucose, HbAlc TG, TC, HDL, creatinine, systolic and diastolic pressure. Muller cells were identified by glutamine synthetase for specific antibodies, and the morphology of the cultured Muller cells in vitro was the same. The frequency of PPARy gene G alleles in.DR patients with glutamine synthetase positive reaction was 5.8%. The genotype of PDR and NPDR patients were dominated by C/C type, and the proportion was 87% and 89.2% respectively. There was no statistical difference between the two groups. Comparison of the main biochemical indexes of different genotypes showed that compared with the DR patients with CC genotype, the UAER level of the patients with G/G or G/C genotype decreased significantly, with statistical difference, but there was no statistical difference between TG, LDL, HDL and HbAlc. Conclusion: the average SFCT of normal Chinese was 271.32 + 35.63gm, and the average SFCT was higher in men of all ages. With the increase of age, SFCT gradually decreased and had a negative correlation with age. The nasal choroid was thinnest. With the increase of DR, the choroidal thickness decreased. The choroid of the PDR patients without DR was significantly thinner than the normal and non DR diabetics, compared with the DME free NPDR mesh, the NPDR choroid with DME's NPDR choroid was significantly thinner.LDL and UAER and DME. Choroidal thickness is negative correlation. Repeated trypsin digestion and culture is a reliable, purified, stable Muller cell in vitro primary culture. The genotype and allele distribution frequency of the.PPARy gene pro12Ala locus and the occurrence of DR, as well as the DR in different stages; but G alleles of the PPAR gamma gene. The gene may reduce the UAER value of DR patients, and may have some protective effect on renal function.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.2;R774.1

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