特异性阻断剂Nec-1对视网膜缺血再灌注损伤模型小鼠坏死性凋亡的影响及作用

发布时间：2018-05-16 02:34

本文选题：特异性阻断剂 + 坏死性凋亡　；参考：《眼科新进展》2017年10期

【摘要】：目的探讨特异性阻断剂Nec-1对视网膜缺血再灌注损伤(retinal ischemia reperfusion injury,RIRI)模型中坏死性凋亡的影响及作用。方法取60只野生型C57小鼠随机分为实验组、对照组及空白组。每组20只。在进行Nec-1对坏死性凋亡影响研究中,空白组15只小鼠不做任何处理,实验组15只小鼠给予玻璃体内注射2μL Nec-1(2mol·L~(-1))预处理,对照组15只小鼠无预处理;预处理4 h后实验组及对照组通过前房灌注法建立RIRI模型;再灌注损伤后3 d,每种检测方法各取5只小鼠分别于取材后行Western blot、免疫荧光定量PCR、免疫荧光染色,检测以下基因mRNA和蛋白的表达变化:IL-1β、IL-6、TNF-α、RIP3、RIP1及Caspase-8。在进行Nec-1对视网膜神经节细胞(retinal ganglion cell,RGC)的影响研究中,实验组、对照组及空白组各5只小鼠纳入荧光金标记。空白组小鼠荧光金标记后不做任何处理。荧光金标记7 d后,实验组小鼠给予玻璃体内注射2μL Nec-1(2 mol·L~(-1))预处理,对照组无预处理。预处理4h后实验组及对照组通过前房灌注法建立RIRI模型。再灌注损伤后3 d,收集视网膜组织行铺片RGC计数研究。结果与对照组相比,实验组RIP3、RIP1的mRNA表达明显降低,差异均有统计学意义(均为P0.001);IL-1β、IL-6、TNF-α的mRNA表达亦均明显降低,差异均有统计学意义(均为P0.001);Caspase-8的表达变化不明显,与对照组相比差异无统计学意义(P=0.654 8)。通过Western blot检测发现,实验组RIP3蛋白表达较对照组明显降低,其变化趋势与RIP3 mRNA水平的变化基本一致。通过视网膜切片免疫荧光染色检测亦发现,实验组RIP3蛋白表达较对照组明显降低。实验组全视网膜铺片中每个视野下荧光金逆行标记RGC数(197.3±3.6)较对照组(107.5±6.1)明显增多,差异有统计学意义(P0.001)。结论实验性RIRI模型中,Nec-1能阻断坏死性凋亡,并显著增加RGC数。
[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- 伪, 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- 伪 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 卤3.6) in the experimental group was significantly higher than that in the control group (107.5 卤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.
【作者单位】：云南省第二人民医院眼科云南省眼科研究所云南省眼科疾病防治研究重点实验室云南省第二人民医院白内障与眼底疾病防治省创新团队;
【基金】：国家自然科学基金资助(编号:81560168)~~
【分类号】：R-332;R774.1

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