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目的通过分析细胞和培养液中的原蛋白转化酶-2,羧基肽酶-E和前神经肽原-Y蛋白的表达水平,研究视网膜神经节细胞-5(retina ganglion cell-5cells,RGC-5)在应激损伤后的神经肽原蛋白加工系统的变化。方法在培养液中加入终浓度为0.1μmol/L的staurosporine并保温24h完成RGC-5的分化;使用氧葡萄糖剥夺(oxygen and glucosedeprivation,OGD)的细胞模型,诱导分化的RGC-5的应激损伤;利用PI和TUNEL染色方法测定急性和慢性应激模型的细胞死亡率;使用Western blot和原蛋白转化酶2的活性测定方法,检测细胞和培养液中的原蛋白转化酶-2,羧基肽酶-E和前神经肽原-Y蛋白表达水平。结果应激反应引起了以剂量依赖性的细胞死亡。随OGD应激剂量的增加,原蛋白转化酶-2和前神经肽原-Y蛋白表达水平逐渐增加,而羧基肽酶原-E逐渐降低。细胞应激损伤后,原蛋白转化酶-2的活性降低。在培养液中,原蛋白转化酶-2和原蛋白转化酶原-2逐渐降低,而羧基肽酶-E,羧基肽酶原-E和前神经肽原-Y渐渐增加。结论应激损伤通过降低原蛋白转化酶-2的活性和原蛋白转化酶原-2的激活,抑制了神经肽原蛋白加工系统,而应激后原蛋白的堆积和羧基肽酶-E细胞外排的增加又反过来加重了细胞的损伤。原蛋白加工系统在应激损伤中起重要作用。
OBJECTIVE: To investigate the expression of retinal ganglion cell-5 cells (RGC-5) in cultured cells and culture media by analyzing the expression of pro-caspase-2, carboxypeptidase-E and procalcitonin- ) Changes in the neuroperoxidin processing system after stress injury. Methods Staurosporine (0.1μmol / L) was added to culture medium and incubated for 24h to complete the differentiation of RGC-5. The cell model of oxygen and glucosedeprivation (OGD) was used to induce the stress injury of differentiated RGC-5 . The cell death rates of acute and chronic stress models were determined by PI and TUNEL staining. The activities of pro-toxin-2 and carboxypeptidase-2 -E and pro-neuropeptide-Y protein expression levels. Results The stress response caused a dose-dependent cell death. With the increase of OGD stress, the expression levels of pro-toase-2 and pro-pro-pro-pro-Y protein increased gradually, while that of carboxypeptidase-E decreased gradually. After the stress of cell stress, the activity of pro-proteinase-2 is reduced. In the culture medium, protoporphyrin-2 and pro-pro-caspase-2 decreased gradually while carboxypeptidase-E, carboxypeptidase-E and procalcitonin-Y increased gradually. CONCLUSIONS: Stress injury inhibits the processing of neuropeptides by reducing the activity of pro-amylase-2 and pro-pro-caspase-2, whereas accumulation of stress progenitor proteins and accumulation of carboxypeptidase-E extracellular In turn, the increase of the row aggravates the cell damage. Raw protein processing systems play an important role in stress injury.