视网膜神经节细胞发育调控网络基因单核苷酸多态性与原发性开角型青光眼的关联研究

发布时间：2018-04-03 10:12

本文选题：原发性开角性青光眼　切入点：视网膜神经节细胞　出处：《青岛大学》2011年博士论文

【摘要】：原发性开角性青光眼(primary open-angle glaucoma, POAG)在西方国家是主要的致盲性眼病之一。20世纪80年代,我国POAG在青光眼中的构成比为8.18%。随着诊断水平的不断提高,其构成比已经上升至19.25%。POAG由于视网膜神经节细胞(retinal ganglion cell, RGCs)缓慢地进行性损害,导致视盘功能改变,并伴有不可逆的视力缺损。很多POAG患者由于发现视力缺损前来就医,但是此时往往错过了最佳的治疗时机,因此预防与早期诊断是成功治疗POAG的关键。由于RGCs功能的改变是导致POAG的主要原因之一,所以阐明RGCs在POAG发病机制中的作用至关重要。同时,POAG具有很大的遗传倾向,且家族史是危险因素之一,因此从发育生物学角度,揭示视网膜、特别是RGCs的发育对POAG的影响,将为阐明POAG的发病机制提供新的思路与理论依据。大量研究表明视网膜以及RGCs的发育与一系列胚胎发育相关的基因时间与空间的表达,彼此之间相互作用,构成的复杂调控网络有关。本研究选择了Pax6、ATOH7、Brn3a、Brn3b、Isl1与Six1基因作为研究的靶基因,采用SNapshot的方法对一高加索人群进行基因分型检测,其中POAG患者为762例,正常对照为464例。基因分型结果表明ATOH7基因的5个SNP在两组间的分布均无统计学差异。Six1 rs 10483727三种基因型在病例组与对照组间的分布具有统计学意义(Genotypic P= 4.07x10-4),两种等位基因在病例组与对照组间的分布具有统计学意义(Allelic P=5.99x10-4),三种基因型在病例组与对照组间的分布趋势具有统计学意义(Trend P= 5.99x10-4)。Six1 rs3783820两种等位基因在病例组与对照组间的分布具有统计学意义(AllelicP= 0.042),三种基因型在病例组与对照组间的分布趋势具有统计学意义(Trend P= 0.042)。Brn3a rs9601092两种等位基因在病例组与对照组间的分布具有统计学意义(Allelic P= 0.022),三种基因型在病例组与对照组间的分布趋势具有统计学意义(Trend P= 0.022)。Pax6 rs3026393与rs2239789三种基因型在病例组与对照组间的分布具有统计学意义(Genotypic P分别为1.09x104与1.14x10-4)。ISL1rs2288468三种基因型在病例组与对照组间的分布具有统计学意义(Genotypic P= 0.039),两种等位基因在病例组与对照组间的分布具有统计学意义(Allelic P=0.010),三种基因型在病例组与对照组间的分布趋势具有统计学意义(Trend P= 0.011)。为进一步验证所选择的基因与POAG的关联性,我们建立了三种诱导RGCs缺失的小鼠模型,分别为视神经损伤模型(Crush model),玻璃体内注射NMDA (N-methyl-d-aspartate)诱导视网膜神经节细胞丢失模型(NMDA model)与视网膜缺血诱导高眼压(IOP model)模型。行荧光实时定量PCR检测建模后不同时间点各相关基因的表达。小鼠视神经crush model术后3天和7天行real time PCR结果表明,与对照组相比,Six1基因在术后3天时表达显著升高,术后7天与术后3天相比其表达有所降低,但仍显著高于对照组。与对照组相比,术后3天Brn3a的表达显著降低,此后其表达继续下降,至术后7天时显著低于对照组。而与对照组相比,术后3天Isll的表达显著降低,至术后7天其表达较术后3天有所升高,但仍显著低于对照组。与对照组相比,Brn3b, Pax6的表达未有显著性差异。 NMDA注射诱导小鼠视神经节细胞死亡后3天和7天行real time PCR结果表明,与对照组相比,Six1基因在术后7天时表达显著升高。术后3天Brn3a的表达显著降低。Pax6的表达在术后3天显著升高,此后其表达下降,至术后7天时低于对照组,但无显著性差异。与对照组相比,术后3天Isll的表达呈上升趋势,但无显著性差异,至术后7天其表达有所降低,显著低于对照组。Brn3b的表达未有显著性差异。IOP诱导小鼠视神经节细胞死亡后1天和5天行real time PCR结果表明,与对照组相比,Six1基因在术后7天时表达显著升高。术后3天Brn3a的表达显著降低。术后3天Pax6的表达显著升高,此后其表达继续上升,至术后7天时显著高于对照组。术后3天Isll的表达呈下降趋势,至术后7天其表达显著高于对照组。综上所述,我们认为在高加索人群中视网膜神经节细胞调控网络基因Pax6, Brn3a, Brn3b, Isl1与Six1可能与原发性开角型青光眼的遗传易感性有关,特别是Six1基因,而未发现与调控RGCs发育与分化的ATOH7基因与POAG的遗传易感性有关。研究结果尚需大样本、多中心的流行病学研究予以验证。
[Abstract]:Primary open-angle glaucoma (primary open-angle glaucoma, POAG) in western countries is the main cause of blindness in.20 century 80s, China's POAG in glaucoma is 8.18%. with diagnostic level increasing, the proportion has risen to 19.25%.POAG due to retinal ganglion cells (retinal ganglion cell, RGCs) slowly damage, leading to disc function change, accompanied by irreversible visual impairment. Many patients with POAG due to the discovery of visual impairment come for medical treatment, but this is often missed the best treatment time, so the prevention and early diagnosis is the key to successful treatment of POAG. Because of changes in the function of RGCs is one of the main causes of POAG so, to clarify RGCs crucial role in the pathogenesis of POAG. At the same time, POAG has great genetic predisposition, and family history is a risk factor, so from The view of developmental biology, to reveal the retina, especially the effect of RGCs on the development of POAG, will provide new ideas and theoretical basis for elucidating the pathogenesis of POAG. A large number of studies show that retinal gene associated with the time and space of RGCs development and a series of embryonic development, the interaction between each other, constitute a complex regulatory network about.
This research chooses Pax6, ATOH7, Brn3a, Brn3b, Isl1 and Six1 gene as target gene research, uses the method of SNapshot to a Caucasian population genotyping detection, including 762 cases of POAG patients, 464 cases of normal control. The genotyping results showed statistically significant distribution of the 5 SNP ATOH7 gene in the two groups were not statistically significant.Six1 RS 10483727 three genotypes in the case group and the control groups (Genotypic P= 4.07x10-4), with statistical significance in the distribution of two kinds of allele between case group and control group (Allelic P =5.99x10-4), with statistical significance in the distribution trend of three genotypes between case group and control group (Trend P= 5.99x10-4) has significant distribution of.Six1 alleles in two rs3783820 between case group and control group (AllelicP= 0.042), three genotypes in patients between the two groups The distribution trend was statistically significant (Trend P= 0.042) with statistical significance.Brn3a distribution of the two alleles of rs9601092 in between case group and control group (Allelic P= 0.022), with statistical significance in the distribution trend of three genotype between case group and control group (Trend P= 0.022) with statistical significance and distribution of.Pax6 rs3026393 the rs2239789 of the three genotypes in the case group and the control group (the Genotypic P 1.09x104 and 1.14x10-4 respectively) were statistically significant in the distribution of.ISL1rs2288468 three genotype between case group and control group (Genotypic P= 0.039), with statistical significance in the distribution of two kinds of allele between case group and control group (Allelic P=0.010), statistically significant distribution trend of three genotypes in the case group and the control groups (Trend P= 0.011).
For the association of POAG gene and further verify the selection, we set up three kinds of induced RGCs deficient mice model respectively for optic nerve injury models (Crush model), intravitreal injection of NMDA (N-methyl-d-aspartate) induced retinal ganglion cell loss model (NMDA model) and retinal ischemia induced by high intraocular pressure (IOP model) model at different time points. The expression of related genes by fluorescence real-time quantitative PCR detection after modeling.
That 3 days and 7 days after real time PCR crush model the mouse optic nerve after operation, compared with the control group, Six1 gene on the 3 postoperative day when the expression was significantly increased after 7 days and 3 days after surgery than its expression decreased, but still significantly higher than the control group. Compared with the control group, the expression of 3 Brn3a days after operation was significantly decreased, then the expression continued to decline, to 7 days after operation was significantly lower than that of control group. Compared with the control group, the expression of Isll in 3 days after surgery was significantly reduced, and after 7 days the expression is 3 days after operation was increased, but still significantly lower than the control group. The expression of Brn3b, and the control group, no significant difference in Pax6.
That 3 days and 7 days after real time PCR results of retinal ganglion cell death of NMDA induced by injection of mice, compared with the control group, Six1 gene expression was significantly increased in the 7 day after surgery. The expression of Brn3a in 3 days after operation was significantly decreased on the 3 day after surgery significantly increased the expression of.Pax6, then the expression decreased to after 7 days when compared with the control group, but no significant difference compared with the control group, after 3 days the expression of Isll increased, but no significant difference to the 7 day after operation, the expression decreased, no significant difference in expression was significantly lower than the control group.Brn3b.IOP induced retinal ganglion cell death the mice after 1 days and 5 days after real time PCR results showed that compared with the control group, Six1 gene expression was significantly increased in the 7 day after surgery. The expression of Brn3a in 3 days after operation. The expression of Pax6 significantly decreased 3 days after surgery increased significantly, then the expression continues to rise, to 7 days after operation was significant The expression of Isll decreased at 3 days after operation, and the expression was significantly higher than that of the control group at the 7 day after operation.
In summary, we believe that in the Caucasian population gonglion cell gene regulatory networks Pax6, Brn3a, Brn3b, Isl1 and Six1 may be related to genetic susceptibility and primary open-angle glaucoma, especially Six1 gene, and found no related genetic susceptibility gene of ATOH7 and POAG and the development and differentiation of RGCs. The results of the study are need a large sample, multi center epidemiological studies to be verified.

【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R775

【共引文献】