本文選題：特異性阻斷劑 + 壞死性凋亡��； 參考：《眼科新進(jìn)展》2017年10期

【摘要】：目的探討特異性阻斷劑Nec-1對(duì)視網(wǎng)膜缺血再灌注損傷(retinal ischemia reperfusion injury,RIRI)模型中壞死性凋亡的影響及作用。方法取60只野生型C57小鼠隨機(jī)分為實(shí)驗(yàn)組、對(duì)照組及空白組。每組20只。在進(jìn)行Nec-1對(duì)壞死性凋亡影響研究中,空白組15只小鼠不做任何處理,實(shí)驗(yàn)組15只小鼠給予玻璃體內(nèi)注射2μL Nec-1(2mol·L~(-1))預(yù)處理,對(duì)照組15只小鼠無預(yù)處理;預(yù)處理4 h后實(shí)驗(yàn)組及對(duì)照組通過前房灌注法建立RIRI模型;再灌注損傷后3 d,每種檢測(cè)方法各取5只小鼠分別于取材后行Western blot、免疫熒光定量PCR、免疫熒光染色,檢測(cè)以下基因mRNA和蛋白的表達(dá)變化:IL-1β、IL-6、TNF-α、RIP3、RIP1及Caspase-8。在進(jìn)行Nec-1對(duì)視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(retinal ganglion cell,RGC)的影響研究中,實(shí)驗(yàn)組、對(duì)照組及空白組各5只小鼠納入熒光金標(biāo)記�？瞻捉M小鼠熒光金標(biāo)記后不做任何處理。熒光金標(biāo)記7 d后,實(shí)驗(yàn)組小鼠給予玻璃體內(nèi)注射2μL Nec-1(2 mol·L~(-1))預(yù)處理,對(duì)照組無預(yù)處理。預(yù)處理4h后實(shí)驗(yàn)組及對(duì)照組通過前房灌注法建立RIRI模型。再灌注損傷后3 d,收集視網(wǎng)膜組織行鋪片RGC計(jì)數(shù)研究。結(jié)果與對(duì)照組相比,實(shí)驗(yàn)組RIP3、RIP1的mRNA表達(dá)明顯降低,差異均有統(tǒng)計(jì)學(xué)意義(均為P0.001);IL-1β、IL-6、TNF-α的mRNA表達(dá)亦均明顯降低,差異均有統(tǒng)計(jì)學(xué)意義(均為P0.001);Caspase-8的表達(dá)變化不明顯,與對(duì)照組相比差異無統(tǒng)計(jì)學(xué)意義(P=0.654 8)。通過Western blot檢測(cè)發(fā)現(xiàn),實(shí)驗(yàn)組RIP3蛋白表達(dá)較對(duì)照組明顯降低,其變化趨勢(shì)與RIP3 mRNA水平的變化基本一致。通過視網(wǎng)膜切片免疫熒光染色檢測(cè)亦發(fā)現(xiàn),實(shí)驗(yàn)組RIP3蛋白表達(dá)較對(duì)照組明顯降低。實(shí)驗(yàn)組全視網(wǎng)膜鋪片中每個(gè)視野下熒光金逆行標(biāo)記RGC數(shù)(197.3±3.6)較對(duì)照組(107.5±6.1)明顯增多,差異有統(tǒng)計(jì)學(xué)意義(P0.001)。結(jié)論實(shí)驗(yàn)性RIRI模型中,Nec-1能阻斷壞死性凋亡,并顯著增加RGC數(shù)。
[Abstract]:Objective to investigate the effect of specific blocker Nec-1 on necrotic apoptosis in retinal ischemia reperfusion injury model. Methods 60 wild-type C 57 mice were randomly divided into three groups: experimental group, control group and blank group. There were 20 rats in each group. In the study of the effect of Nec-1 on necrotic apoptosis, 15 mice in the blank group were treated without any treatment, 15 mice in the experimental group were pretreated with intravitreous injection of 2 渭 L Nec-1(2mol LP-1), and 15 mice in the control group were not pretreated. After pretreatment for 4 h, the experimental group and the control group established the RIRI model by anterior chamber perfusion, and on the 3rd day after reperfusion injury, 5 mice were taken from each method to perform Western blot, immunofluorescence quantitative Western and immunofluorescence staining respectively. To detect the expression of mRNA and protein in the following genes: IL-1 尾, IL-6, TNF- 偽, RIP3, RIP1 and Caspase-8. In the study of the effect of Nec-1 on retinal ganglion cell (RGC) in retinal ganglion cells, 5 mice in the experimental group, 5 mice in the control group and 5 mice in the blank group were labeled with fluorescent gold. The mice in blank group were labeled with fluorescent gold without any treatment. The mice in the experimental group were pretreated with intravitreous injection of 2 渭 L Nec-1(2 mol LP-1 after 7 days of fluorescent gold labeling, while the control group was not pretreated. RIRI model was established by anterior chamber perfusion in experimental group and control group 4 h after pretreatment. RGC count of retinal tissue was collected 3 days after reperfusion injury. Results compared with the control group, the mRNA expression of RIP3 and RIP1 in the experimental group was significantly lower than that in the control group, and the difference was statistically significant (both P0.001, IL-1 尾 and IL-6TNF- 偽 mRNA expression was significantly lower than that in the control group, and there was no significant difference in the expression of Caspase-8 between the two groups. There was no significant difference between the control group and the control group. Western blot analysis showed that the expression of RIP3 protein in the experimental group was significantly lower than that in the control group, and the change trend was consistent with the change of RIP3 mRNA level. The expression of RIP3 protein in the experimental group was significantly lower than that in the control group. The number of fluorescent gold retrograde labeled RGC (197.3 鹵3.6) in the experimental group was significantly higher than that in the control group (107.5 鹵6.1), and the difference was statistically significant (P 0.001). Conclusion Nec-1 can block necrotic apoptosis and increase the number of RGC in experimental RIRI model.
【作者單位】： 云南省第二人民醫(yī)院眼科云南省眼科研究所云南省眼科疾病防治研究重點(diǎn)實(shí)驗(yàn)室云南省第二人民醫(yī)院白內(nèi)障與眼底疾病防治省創(chuàng)新團(tuán)隊(duì);
【基金】：國(guó)家自然科學(xué)基金資助(編號(hào):81560168)~~
【分類號(hào)】：R-332;R774.1

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