CAJ | 학술논문

目的观察实验性视网膜脱离后光感受器细胞坏死性凋亡的形态学特点,初步探讨其发生机制.方法雄性健康的Sprague-Dawley大鼠60只,右眼视网膜下注入1％透明质酸纳50μl建立视网膜脱离模型作为实验组,左眼不作处理为对照组.建模后3d,采用电子显微镜观察光感受器细胞的死亡方式及形态学特点.建模后3d,提取大鼠视网膜组织,采用蛋白质免疫印迹法检测裂解的半胱氨酸蛋白酶(Caspase)8的相对蛋白表达;采用免疫沉淀法检测磷酸化受体相互作用蛋白1(p-RIP1)的相对蛋白表达.结果电子显微镜观察发现,视网膜脱离后光感受器细胞的死亡方式主要为凋亡和坏死.同时还可观察到光感受器细胞的坏死性凋亡,其形态学与坏死细胞类似.表现为细胞肿胀,质膜不完整、破裂,染色质浓集和边聚,且伴有明显的自噬泡和空泡.蛋白质免疫印迹法检测结果显示,实验组、对照组裂解的Caspase 8蛋白相对表达分别为0.78±0.03、0.06±0.01,实验组裂解的Caspase 8蛋白相对表达较对照组明显增加.两组裂解的Caspase 8蛋白相对表达比较,差异有统计学意义(F=4 023.21,P＜0.05).免疫沉淀法检测结果显示,实验组、对照组p-RIP1蛋白相对表达分别为0.23±0.03、0.14±0.02,实验组p-RIP1蛋白相对表达较对照组明显增加.两组p-RIP1蛋白相对表达比较,差异有统计学意义(F=56.44,P＜0.05).结论实验性视网膜脱离后光感受器细胞坏死性凋亡表现为细胞肿胀,质膜不完整、破裂,染色质浓集和边聚,且伴有明显的自噬泡和空泡;其发生机制与p-RIP1表达增加有关.
목적 관찰실험성시망막탈리후광감수기세포배사성조망적형태학특점,초보탐토기발생궤제.방법 웅성건강적Sprague-Dawley대서60지,우안시망막하주입1％투명질산납50μl건립시망막탈리모형작위실험조,좌안불작처리위대조조.건모후3d,채용전자현미경관찰광감수기세포적사망방식급형태학특점.건모후3d,제취대서시망막조직,채용단백질면역인적법검측렬해적반광안산단백매(Caspase)8적상대단백표체;채용면역침정법검측린산화수체상호작용단백1(p-RIP1)적상대단백표체.결과 전자현미경관찰발현,시망막탈리후광감수기세포적사망방식주요위조망화배사.동시환가관찰도광감수기세포적배사성조망,기형태학여배사세포유사.표현위세포종창,질막불완정、파렬,염색질농집화변취,차반유명현적자서포화공포.단백질면역인적법검측결과현시,실험조、대조조렬해적Caspase 8단백상대표체분별위0.78±0.03、0.06±0.01,실험조렬해적Caspase 8단백상대표체교대조조명현증가.량조렬해적Caspase 8단백상대표체비교,차이유통계학의의(F=4 023.21,P＜0.05).면역침정법검측결과현시,실험조、대조조p-RIP1단백상대표체분별위0.23±0.03、0.14±0.02,실험조p-RIP1단백상대표체교대조조명현증가.량조p-RIP1단백상대표체비교,차이유통계학의의(F=56.44,P＜0.05).결론 실험성시망막탈리후광감수기세포배사성조망표현위세포종창,질막불완정、파렬,염색질농집화변취,차반유명현적자서포화공포;기발생궤제여p-RIP1표체증가유관.
Objective To observe the morphological characteristics of photoreceptor necroptosis in experimental retinal detachment,and explore the mechanism.Methods A total of 60 Sprague-Dawley male rats were included in this study.Retinal detachment were induced in the right eyes with 1％ sodium hyaluronate (50 μl) injection (experimental group),while the left eyes received no treatment (control group).At 3 days after modeling,the morphological characteristics of photoreceptor cell were observed by electron microscopy.Cleaved Caspase 8 and phosphorylation receptor-interacting protein 1 (p-RIP1) were measured by Western blot and immunoprecipitation.Results At 3 days after modeling,photoreceptor necroptosis showed the following morphological features:chromatin condensation,severe vacuolation,early loss of plasma membrane integrity,and many autophagosomes.Western blot showed that the protein expression of cleaved Caspase 8 were 0.78 ± 0.03,0.06 ± 0.01 in experimental group and control group respectively,which was significantly different (F=4 023.21,P＜0.05).Immunoprecipitation showed that the protein expression of p-RIP1 were 0.23±0.03,0.14±0.02 in experimental group and control group respectively,which was significantly different (F =56.44,P ＜ 0.05).Conclusions Photoreceptor necroptosis showed chromatin condensation,severe vacuolation,early loss of plasma membrane integrity,and many autophagosomes.Necroptosis activation was associated with the increase of RIP1 phosphorylation.