本文選題：原發(fā)性開(kāi)角性青光眼　切入點(diǎn)：視網(wǎng)膜神經(jīng)節(jié)細(xì)胞　出處：《青島大學(xué)》2011年博士論文

【摘要】：原發(fā)性開(kāi)角性青光眼(primary open-angle glaucoma, POAG)在西方國(guó)家是主要的致盲性眼病之一。20世紀(jì)80年代,我國(guó)POAG在青光眼中的構(gòu)成比為8.18%。隨著診斷水平的不斷提高,其構(gòu)成比已經(jīng)上升至19.25%。POAG由于視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(retinal ganglion cell, RGCs)緩慢地進(jìn)行性損害,導(dǎo)致視盤(pán)功能改變,并伴有不可逆的視力缺損。很多POAG患者由于發(fā)現(xiàn)視力缺損前來(lái)就醫(yī),但是此時(shí)往往錯(cuò)過(guò)了最佳的治療時(shí)機(jī),因此預(yù)防與早期診斷是成功治療POAG的關(guān)鍵。由于RGCs功能的改變是導(dǎo)致POAG的主要原因之一,所以闡明RGCs在POAG發(fā)病機(jī)制中的作用至關(guān)重要。同時(shí),POAG具有很大的遺傳傾向,且家族史是危險(xiǎn)因素之一,因此從發(fā)育生物學(xué)角度,揭示視網(wǎng)膜、特別是RGCs的發(fā)育對(duì)POAG的影響,將為闡明POAG的發(fā)病機(jī)制提供新的思路與理論依據(jù)。大量研究表明視網(wǎng)膜以及RGCs的發(fā)育與一系列胚胎發(fā)育相關(guān)的基因時(shí)間與空間的表達(dá),彼此之間相互作用,構(gòu)成的復(fù)雜調(diào)控網(wǎng)絡(luò)有關(guān)。 本研究選擇了Pax6、ATOH7、Brn3a、Brn3b、Isl1與Six1基因作為研究的靶基因,采用SNapshot的方法對(duì)一高加索人群進(jìn)行基因分型檢測(cè),其中POAG患者為762例,正常對(duì)照為464例。基因分型結(jié)果表明ATOH7基因的5個(gè)SNP在兩組間的分布均無(wú)統(tǒng)計(jì)學(xué)差異。Six1 rs 10483727三種基因型在病例組與對(duì)照組間的分布具有統(tǒng)計(jì)學(xué)意義(Genotypic P= 4.07x10-4),兩種等位基因在病例組與對(duì)照組間的分布具有統(tǒng)計(jì)學(xué)意義(Allelic P=5.99x10-4),三種基因型在病例組與對(duì)照組間的分布趨勢(shì)具有統(tǒng)計(jì)學(xué)意義(Trend P= 5.99x10-4)。Six1 rs3783820兩種等位基因在病例組與對(duì)照組間的分布具有統(tǒng)計(jì)學(xué)意義(AllelicP= 0.042),三種基因型在病例組與對(duì)照組間的分布趨勢(shì)具有統(tǒng)計(jì)學(xué)意義(Trend P= 0.042)。Brn3a rs9601092兩種等位基因在病例組與對(duì)照組間的分布具有統(tǒng)計(jì)學(xué)意義(Allelic P= 0.022),三種基因型在病例組與對(duì)照組間的分布趨勢(shì)具有統(tǒng)計(jì)學(xué)意義(Trend P= 0.022)。Pax6 rs3026393與rs2239789三種基因型在病例組與對(duì)照組間的分布具有統(tǒng)計(jì)學(xué)意義(Genotypic P分別為1.09x104與1.14x10-4)。ISL1rs2288468三種基因型在病例組與對(duì)照組間的分布具有統(tǒng)計(jì)學(xué)意義(Genotypic P= 0.039),兩種等位基因在病例組與對(duì)照組間的分布具有統(tǒng)計(jì)學(xué)意義(Allelic P=0.010),三種基因型在病例組與對(duì)照組間的分布趨勢(shì)具有統(tǒng)計(jì)學(xué)意義(Trend P= 0.011)。 為進(jìn)一步驗(yàn)證所選擇的基因與POAG的關(guān)聯(lián)性,我們建立了三種誘導(dǎo)RGCs缺失的小鼠模型,分別為視神經(jīng)損傷模型(Crush model),玻璃體內(nèi)注射N(xiāo)MDA (N-methyl-d-aspartate)誘導(dǎo)視網(wǎng)膜神經(jīng)節(jié)細(xì)胞丟失模型(NMDA model)與視網(wǎng)膜缺血誘導(dǎo)高眼壓(IOP model)模型。行熒光實(shí)時(shí)定量PCR檢測(cè)建模后不同時(shí)間點(diǎn)各相關(guān)基因的表達(dá)。 小鼠視神經(jīng)crush model術(shù)后3天和7天行real time PCR結(jié)果表明,與對(duì)照組相比,Six1基因在術(shù)后3天時(shí)表達(dá)顯著升高,術(shù)后7天與術(shù)后3天相比其表達(dá)有所降低,但仍顯著高于對(duì)照組。與對(duì)照組相比,術(shù)后3天Brn3a的表達(dá)顯著降低,此后其表達(dá)繼續(xù)下降,至術(shù)后7天時(shí)顯著低于對(duì)照組。而與對(duì)照組相比,術(shù)后3天Isll的表達(dá)顯著降低,至術(shù)后7天其表達(dá)較術(shù)后3天有所升高,但仍顯著低于對(duì)照組。與對(duì)照組相比,Brn3b, Pax6的表達(dá)未有顯著性差異。 NMDA注射誘導(dǎo)小鼠視神經(jīng)節(jié)細(xì)胞死亡后3天和7天行real time PCR結(jié)果表明,與對(duì)照組相比,Six1基因在術(shù)后7天時(shí)表達(dá)顯著升高。術(shù)后3天Brn3a的表達(dá)顯著降低。Pax6的表達(dá)在術(shù)后3天顯著升高,此后其表達(dá)下降,至術(shù)后7天時(shí)低于對(duì)照組,但無(wú)顯著性差異。與對(duì)照組相比,術(shù)后3天Isll的表達(dá)呈上升趨勢(shì),但無(wú)顯著性差異,至術(shù)后7天其表達(dá)有所降低,顯著低于對(duì)照組。Brn3b的表達(dá)未有顯著性差異。IOP誘導(dǎo)小鼠視神經(jīng)節(jié)細(xì)胞死亡后1天和5天行real time PCR結(jié)果表明,與對(duì)照組相比,Six1基因在術(shù)后7天時(shí)表達(dá)顯著升高。術(shù)后3天Brn3a的表達(dá)顯著降低。術(shù)后3天Pax6的表達(dá)顯著升高,此后其表達(dá)繼續(xù)上升,至術(shù)后7天時(shí)顯著高于對(duì)照組。術(shù)后3天Isll的表達(dá)呈下降趨勢(shì),至術(shù)后7天其表達(dá)顯著高于對(duì)照組。 綜上所述,我們認(rèn)為在高加索人群中視網(wǎng)膜神經(jīng)節(jié)細(xì)胞調(diào)控網(wǎng)絡(luò)基因Pax6, Brn3a, Brn3b, Isl1與Six1可能與原發(fā)性開(kāi)角型青光眼的遺傳易感性有關(guān),特別是Six1基因,而未發(fā)現(xiàn)與調(diào)控RGCs發(fā)育與分化的ATOH7基因與POAG的遺傳易感性有關(guān)。研究結(jié)果尚需大樣本、多中心的流行病學(xué)研究予以驗(yàn)證。
[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.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R775

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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3 李金瑛,，

本文編號(hào)：1704746