【摘要】：目的:氧化應(yīng)激引起線粒體功能障礙并最終導(dǎo)致視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的死亡。白藜蘆醇具有抗炎抗氧化應(yīng)激作用,并且通過(guò)激活SIRT1來(lái)發(fā)揮其保護(hù)作用。但是,白藜蘆醇是否可以通過(guò)激活SIRT1來(lái)保護(hù)線粒體處于未知階段。目前關(guān)于Sirtuins的研究越來(lái)越多,但是在眼科領(lǐng)域還處于未知階段。因此本研究主要探討1:Sirtuin家族在大鼠急性缺血損傷視網(wǎng)膜和視神經(jīng)中的表達(dá)情況。2.在視網(wǎng)膜缺血損傷動(dòng)物模型中,白藜蘆醇對(duì)線粒體的保護(hù)作用并且是否通過(guò)SIRT1來(lái)介導(dǎo)的。方法:(1)選取成年大鼠,隨機(jī)選取右眼做急性短暫性視網(wǎng)膜缺血模型,左眼作為對(duì)照組,分別于1d、3d、7d提取視網(wǎng)膜組織蛋白檢測(cè)OPA1、Drp1、SIRT1、Sirtuins蛋白表達(dá)。(2)取正常成年大鼠視網(wǎng)膜,通過(guò)免疫熒光檢測(cè)SIRT1和OPA1在視網(wǎng)膜表達(dá)以及檢測(cè)Sirtuins在視網(wǎng)膜和視神經(jīng)的表達(dá)。(3)大鼠急性視網(wǎng)膜缺血損傷后立即給予白藜蘆醇處理后7d提取視網(wǎng)膜組織檢測(cè)OPA1、Drp1、SIRT1蛋白表達(dá)。(4)定量PCR檢測(cè)opa1、drp1、sirt1和Sirtuins基因的表達(dá)。(5)通過(guò)免疫沉淀檢測(cè)SIRT1和OPA1的相關(guān)性。結(jié)果:(1)正常大鼠視網(wǎng)膜中,Sirtuins、OPA1、SIRT1和Sirtuins陽(yáng)性表達(dá)主要在視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層、內(nèi)核層、外核層,其中OPA1和SIRT1共同在視網(wǎng)膜神經(jīng)節(jié)細(xì)胞細(xì)胞質(zhì)表達(dá)陽(yáng)性表達(dá)最明顯。(2)與正常組0d相比,Sirtuin家族變化不同。與正常組0d相比,視網(wǎng)膜缺血損傷后1d,SIRT1的表達(dá)基本不變,視網(wǎng)膜缺血損傷后3d、7d后SIRT1的表達(dá)下降,7d明顯下降(*p0.05);與正常組0d相比,視網(wǎng)膜缺血損傷后1d,OPA1的表達(dá)升高,視網(wǎng)膜缺血損傷后3d、7d后OPA1的表達(dá)又下降,7d明顯下降(*p0.05);與正常組0d相比,視網(wǎng)膜缺血損傷后1d,Drp1的表達(dá)下降(*p0.05),視網(wǎng)膜缺血損傷后3d,Drp1表達(dá)相比1d又升高(*p0.05),7d后Drp1的表達(dá)相比0d明顯下降(**p0.01)。(3)與正常組0d相比,視網(wǎng)膜缺血損傷后7d OPA1表達(dá)明顯下降(*p0.05);給予RSV處理后,OPA1的表達(dá)又升高且升高明顯(*p0.05);與正常組0d相比,視網(wǎng)膜缺血損傷后7d Drp1表達(dá)明顯下降(*p0.05);給予RSV處理后,Drp1表達(dá)相對(duì)0d下降(*p0.05),并且相對(duì)7d Drp1繼續(xù)下降,但表達(dá)無(wú)明顯差異(P0.05);與正常組0d相比,視網(wǎng)膜缺血損傷后7d SIRT1表達(dá)明顯下降(*p0.05);給予RSV處理后,SIRT1的表達(dá)又升高且升高明顯(*p0.05)。(4)與正常組0d相比,Sirtuin家族基因表達(dá)變化不同。與正常組0d相比,視網(wǎng)膜缺血損傷后7d opa1基因表達(dá)明顯下降(*p0.05);給予RSV處理后,opa1基因表達(dá)又升高且升高明顯(*p0.05);與正常組0d相比,視網(wǎng)膜缺血損傷后7d drp1基因表達(dá)明顯下降(*p0.05);給予RSV處理后,drp1基因表達(dá)相對(duì)7d又明顯升高(*p0.05)。(5)SIRT1和OPA1沒(méi)有直接起作用。結(jié)論:(1)Sirtuin家族在大鼠急性缺血損傷視網(wǎng)膜和視神經(jīng)表達(dá)不同。(2)視網(wǎng)膜急性缺血損傷后,SIRT1呈下降趨勢(shì),OPA1先升高再下降、Drp1表達(dá)和OPA1相反。(3)白藜蘆醇對(duì)SIRT1有保護(hù)作用,同時(shí)保護(hù)線粒體,促進(jìn)線粒體的融合、抑制其裂解。
[Abstract]:Objective: oxidative stress causes mitochondrial dysfunction and eventually leads to the death of retinal ganglion cells. Resveratrol has anti-inflammatory and antioxidant stress and plays a protective role by activating SIRT1. However, whether resveratrol can protect mitochondria at an unknown stage by activating SIRT1. At present, there are more and more research on Sirtuins, but it is still in an unknown stage in the field of ophthalmology. Therefore, the purpose of this study was to investigate the expression of 1:Sirtuin family in the retina and optic nerve of rats with acute ischemic injury. 2. In the animal model of retinal ischemic injury, resveratrol protects mitochondria and is mediated by SIRT1. Methods: (1) Adult rats were randomly selected to make acute transient retinal ischemia model in the right eye, and the left eye was used as the control group. The retinal tissue protein was extracted on the 1st day, the 3rd day, and the OPA1,Drp1,SIRT1, was detected on the 7th day. Expression of Sirtuins protein. (2) the retinal tissue of normal adult rats was taken from the retinal tissue of normal adult rats. The expression of SIRT1 and OPA1 in the retina and the expression of Sirtuins in the retinal and optic nerve were detected by immunofluorescence. (3) the retinal tissue was extracted to detect OPA1,Drp1, 7 days after acute retinal ischemia and injury. (3) 7 days after resveratrol treatment, the retinal tissue was extracted to detect OPA1,Drp1,. (4) quantitative PCR was used to detect the expression of opa1,drp1,sirt1 and Sirtuins genes. (5) the correlation between SIRT1 and OPA1 was detected by immunoprecipitation. Results: (1) the positive expression of Sirtuins,OPA1,SIRT1 and Sirtuins was mainly in the retinal ganglion cell layer, inner nuclear layer and outer nuclear layer of normal rats. The positive expression of OPA1 and SIRT1 in the cytoplasm of retinal ganglion was the most obvious. (2) the change of Sirtuin family was different from that of normal group on day 0. Compared with the normal group, the expression of SIRT1 remained unchanged on the 1st day after retinal ischemic injury, and the expression of SIRT1 decreased on the 3rd day and 7th day after retinal ischemic injury, and decreased significantly on the 7th day after retinal ischemic injury (* p0.05). Compared with the normal group, the expression of OPA1 increased on the 1st day after retinal ischemic injury, and the expression of OPA1 decreased again on the 3rd day after retinal ischemic injury, and decreased significantly on the 7th day after retinal ischemic injury (* p0.05). Compared with the normal group, the expression of Drp1 decreased on the 1st day after retinal ischemic injury (* p0.05), and increased on the 3rd day after retinal ischemic injury (* p0.05). Compared with the normal group, the expression of Drp1 decreased significantly on the 7th day (* p0.01). (3), and the expression of OPA1 on the 7th day after retinal ischemia injury was significantly lower than that of the normal group (* p0.05). After treatment with RSV, the expression of OPA1 increased significantly (* p0.05), and the expression of Drp1 decreased significantly on the 7th day after retinal ischemia injury compared with the normal group (* p0.05). After treatment with RSV, the expression of Drp1 decreased relative to 0 days (* p0.05), and the expression of Drp1 continued to decrease compared with 7 days, but there was no significant difference in the expression of Drp1 (P 0.05). Compared with the normal group on the 0th day, the expression of SIRT1 decreased significantly on the 7th day after retinal ischemic injury (* p0.05). After treatment with RSV, the expression of SIRT1 increased and increased significantly (* p0.05). (4), and the expression of Sirtuin family gene was different from that of normal group on day 0. Compared with the normal group, the expression of opa1 gene decreased significantly on the 7th day after retinal ischemia injury (* p0.05), and the expression of opa1 gene increased significantly after RSV treatment (* p0.05). Compared with the normal group, the expression of drp1 gene decreased significantly on the 7th day after retinal ischemia injury (* p0.05), and the expression of drp1 gene increased significantly on the 7th day after RSV treatment (* p0.05). (5). SIRT1 and OPA1 did not play a direct role. Conclusion: (1) the expression of Sirtuin family in retinal and optic nerve of rats with acute ischemic injury is different. (2) after acute ischemic injury of retinal, SIRT1 tends to decrease, OPA1 increases at first and then decreases. The expression of Drp1 is opposite to that of OPA1. (3) resveratrol has protective effect on SIRT1, at the same time, it can protect mitochondria, promote the fusion of mitochondria and inhibit its fragmentation.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R775

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7 郝，

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