抑制自噬在视网膜光感受器细胞光损伤中的保护作用

发布时间：2018-07-21 10:10

【摘要】：自噬是一种保守的细胞自我退化的过程,在细胞生长关键时期如发育阶段或营养缺乏时常常对细胞发挥保护作用。然而,有时自噬也参与细胞凋亡进程。光感受器细胞是唯一直接暴露在光照下将光刺激转换为视觉信号的神经元细胞。但过强的光照射对视网膜仍具有细胞毒性作用。至今为止,视网膜光损伤的确切机制尚不明确,并且临床上仍无有效治疗方案,特别是自噬在光诱导感光细胞凋亡中的作用机制有待进一步探讨。 1.光照对诱导661W细胞产生自噬的作用目的：通过不同的自噬检测方法,观察2600Lux可见光照射对661W细胞自噬的影响。方法：(1)利用2600Lux可见光处理传代培养后的661W细胞株,建立光感受器细胞光损伤模型。(2)利用WeStesrn blotting检测分析自噬标志性蛋白LC3在细胞中的表达水平,以及利用电子显微镜对自噬体形态学变化进行观察。(3)应用3-MA和LY294002两种不同的自噬抑制剂对661W细胞进行预处理后,通过PI染色进行细胞死亡百分比定量评估,观察抑制自噬对661W细胞光损伤保护作用的影响。结果：(1)在光强度26001ux照射1-2d的661W细胞中LC3II表达水平明显增强,实验组细胞中LC3II/LC3I的比例明显高于对照组细胞,其差异有统计学意义(P0.001)。经过2天光照后,661w细胞表现出典型的自噬特性,一些胞内物质如细胞器被包绕在双层膜囊内,成为自噬小体。(2)应用10-30μM的LY294002光照前30分钟对细胞进行预处理,细胞死亡比例从63%下降至20%,其最佳浓度在20μM左右；661W细胞与2.5-7.5mM的3-MA预孵化,细胞死亡率从63%减少至30%,在5mM显示出最好的保护作用。其差异有统计学意义(P0.001)。结论：光照是诱导661W细胞发生自噬的关键,而且阻断自噬可以有效地保护感光细胞免受损害。 2. MAPK信号途径对光诱导661W细胞自噬的影响目的：研究在2600Lux可见光照射条件下,MAPK信号途径对661W细胞活性的影响,探讨自噬在光诱导感光细胞凋亡中的作用机制。方法：(1)利用2600Lux可见光处理传代培养后的661W细胞株,建立光感受器细胞光损伤模型。(2)利用Western blotting检测ERK/磷酸化ERK, JNK/磷酸化JNK, p38/磷酸化p38的表达水平,通过PI染色对ERK抑制剂PD980519, JNK抑制剂SP600125和P38抑制剂SB203580预处理后的661W细胞进行细胞死亡百分比定量评估。(3)在指定时间内收集细胞裂解,利用Western blotting检测ERK和磷酸化ERK后LC3II和LC3I表达水平的变化,以及通过LysoTracker染色观察自噬小体内溶酶体生成情况。结果：(1)收集细胞裂解,对ERK/磷酸化ERK, JNK/磷酸化JNK, p38/磷酸化p38的表达水平进行蛋白免疫印迹分析,实验组(磷酸化组)扫描蛋白条带光密度测量强度明显增加,对照组(非磷酸化)只略有增加,其差异有统计学意义(P0.001)。用40-80μM的PD980519对细进行预处理,光诱导的细胞死亡比例从61%减少至约30%,而SP600125或SB203580对细胞免受光损伤的保护作用效果不显著,差异有统计学意义(P0.001)。(2)通过Western blot检测发现,用40μPD980519在光照30分钟前处理细胞,逆转了P-ERK和ERK的上调。定量分析结果表明,在PD980519预处理组非磷酸化和磷酸化形式的ERK水平均显著减少。而且光照2天后用40μPD980519处理组降低了LC3II/LC3I比率的上调。通过Lyso Tracker染色发现：用40μMPD980519预处理即使在光照下也明显抑制了细胞溶酶体的形成。差异均具有统计学意义(P0.001)。结论：可见光照射可以激活活化蛋白酶(MAPK)通路导致661W细胞发生自噬,在激活的MAPK信号下游通路中,是ERK通路而不是JNK或P-38在光诱导的细胞死亡机制中发挥了关键作用。通过阻断ERK途径能有效地抑制光诱导的661W细胞发生自噬。本研究的发现可能在视网膜光损伤的治疗中具有潜在应用价值。
[Abstract]:Autophagy is a conservative process of self degradation and often protects cells at critical stages of cell growth, such as developmental or nutritional deficiency. However, autophagy sometimes participates in the process of apoptosis. Photoreceptor cells are the only neuron cells that directly expose light stimulation to visual signals under light. Too strong light irradiation still has cytotoxic effects on the retina. Up to now, the exact mechanism of retinal light damage is not clear, and there is still no effective treatment in clinic, especially the mechanism of autophagy in photoreceptor cell apoptosis.
The effect of 1. light on the induction of autophagy in 661W cells
Objective: To observe the effect of 2600Lux visible light irradiation on autophagy of 661W cells by different autophagy methods. Methods: (1) 661W cell lines were treated by 2600Lux visible light treatment, and the photoreceptor cell light damage model was established. (2) the expression of autophagic marker protein LC3 in cell was detected by WeStesrn blotting. 3-MA and LY294002 were used to observe the morphological changes of autophago. (3) after pre treatment of 661W cells with two different autophagic inhibitors, the percentage of cell death was evaluated by PI staining, and the effect of autophagy on the protection of light damage of 661W cells was observed. Results: (1) in light intensity 26 The expression level of LC3II in 661W cells irradiated by 001ux was obviously enhanced. The proportion of LC3II/LC3I in the experimental group was significantly higher than that of the control group. The difference was statistically significant (P0.001). After 2 days of illumination, the 661W cells showed typical autophagy, and some intracellular substances such as the organelles were wrapped around the double layer membrane and became autophagic bodies. (2) the cells were pretreated with 10-30 M LY294002 light before 30 minutes, the proportion of cell death decreased from 63% to 20%, and the optimum concentration was around 20 M; 661W cells incubated with 2.5-7.5mM 3-MA and the cell mortality decreased from 63% to 30%. The difference was statistically significant (P0.001). Conclusion: light (P0.001). Conclusion: light (P0.001). Irradiation is the key to induce autophagy in 661W cells, and blocking autophagy can effectively protect photoreceptors from damage.
Effect of 2. MAPK signaling pathway on autophagy of 661W cells induced by light
Objective: To investigate the effect of MAPK signal pathway on the activity of 661W cells under 2600Lux visible light, and to explore the mechanism of autophagy in photoreceptor cell apoptosis. Methods: (1) using 2600Lux visible light to treat 661W cell lines after subculture, and to establish light damage model of photoreceptor cells. (2) Western blotting detection ERK/ phosphorylated ERK, JNK/ phosphorylated JNK, p38/ phosphorylated p38 expression level. The percentage of cell death of ERK inhibitor PD980519, JNK inhibitor SP600125 and P38 inhibitor pretreatment were evaluated by PI staining. (3) cell lysis was collected at a specified time, and the cells were detected and phosphorylated. The changes in the expression level of LC3II and LC3I after RK, as well as the formation of lysosomes in the small body of autophagy by LysoTracker staining. Results: (1) collect cell lysis, analyze the expression level of ERK/ phosphorylated ERK, JNK/ phosphorylated JNK, p38/ phosphorylated p38, and the test group (phosphorylation group) scanning protein strip light density measurement The strength of the control group (non phosphorylation) was only slightly increased, and the difference was statistically significant (P0.001). The proportion of light induced cell death was reduced from 61% to about 30% with 40-80 M PD980519, while the protective effect of SP600125 or SB203580 on cell protection from light damage was not significant, the difference was statistically significant (P0.001) (2) through Western blot detection, it was found that the up regulation of P-ERK and ERK was reversed with 40 mu PD980519 before light irradiation for 30 minutes. The quantitative analysis showed that the ERK level in the form of non phosphorylation and phosphorylation in the PD980519 preconditioning group decreased significantly. And the up regulation of LC3II/LC3I ratio was reduced by the 40 u PD980519 treatment group after 2 days of light. It was found by Lyso Tracker staining that the formation of lysosomes in cells was obviously inhibited even under light. The difference was statistically significant (P0.001). Conclusion: light irradiation can activate the activation protease (MAPK) pathway to cause 661W cells to occur autophagy, and in the downstream pathway of the activated MAPK signal, it is ERK pass. Roads, not JNK or P-38, play a key role in the mechanism of light induced cell death. By blocking the ERK pathway, autophagy can be effectively inhibited by light induced 661W cells.
The findings of this study may have potential application in the treatment of retinal photodamage.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R774

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