依托咪酯对机械性损伤的体外培养视网膜神经节细胞的保护作用

Protective effect of etomidate on cultured retinal ganglion cells with mechanical injury in vitro

目的观察依托咪酯(ET)对机械性损伤的体外培养视网膜神经节细胞(RGC)的保护作用。方法体外培养新生Sprague-Dawley大鼠RGC,采用Thy1.1和微管关联蛋白2对其进行免疫荧光双标鉴定。培养的原代细胞随机分为对照组、RGC划伤组、ET低剂量组(1μmol/L)、ET中剂量组(5μmol/L)、ET高剂量组(10μmol/L)。RGC划伤组和不同浓度ET组采用虹膜刀划伤培养细胞,建立RGC机械性损伤模型。建模后7 d,细胞计数试剂盒-8增生分析法检测各组RGC存活率;膜联蛋白Ⅴ/碘化丙啶双染色法检测各组RGC凋亡率。多组间比较采用单因素方差分析;组间两两比较采用最小显著差法检验。结果RGC划伤组、ET低剂量组、ET中剂量组、ET高剂量组的RGC存活率分别为(72.60±2.97)%、(73.73±1.14)%、(79.19±1.79)%、(83.88±0.94)%;对照组、RGC划伤组、ET低剂量组、ET中剂量组、ET高剂量组的RGC凋亡率分别为(5.08±0.17)%、(18.67±1.24)%、(17.96±0.74)%、(15.11±0.56)%、(11.67±1.32)%。组间RGC存活率比较:与RGC划伤组相比,ET低剂量组、ET中剂量组、ET高剂量组的细胞存活率增高,RGC划伤组与ET低剂量组之间的差异无统计学意义(P=0.728);RGC划伤组与ET中剂量组、ET高剂量组之间的差异均有统计学意义(P<0.001);ET中剂量组与ET高剂量组之间的差异有统计学意义(P=0.002)。组间RGC凋亡率比较:RGC划伤组凋亡率较对照组显著升高,差异有统计学意义(P<0.001);与RGC划伤组比较,ET低剂量组、ET中剂量组、ET高剂量组的细胞凋亡率降低,RGC划伤组与ET低剂量组之间的差异无统计学意义(P=0.869);R G C划伤组与E T中剂量组、E T高剂量组之间的差异均有统计学意义(P<0.05);ET中剂量组与ET高剂量组之间的差异有统计学意义(P=0.007)。结论ET对机械性损伤的体外培养RGC具有神经保护作用,且保护作用在一定范围内随ET剂量的增长而增强。

Objective To observe the protective effect of etomidate(ET)on cultured retinal ganglion cells(RGC)with mechanical injury in vitro.Methods New Sprague-Dawley rat RGC was cultured in vitro and identified by double immunofluorescent labeling of Thy1.1 and microtubule associated protein 2.The cultured primary cells were randomly divided into control group,RGC scratch group,ET low dose group(1μmol/L),ET medium dose group(5μmol/L)and ET high dose group(10μmol/L).The RGC mechanical injury model was established by using iris knife to culture cells in RGC scratch group and ET group with different concentration.Seven days after modeling,the RGC survival rate of each group was detected by cell count Kit 8 proliferation assay.The apoptosis rate of RGC was detected by AnnexinⅤ/propyl iodide double staining.Single factor analysis of variance was used to compare the groups.The pairwise comparison between groups was tested by the least significant difference method.Results The survival rates of RGC in RGC scratch group,ET low dose group,ET medium dose group and ET high dose group were(72.60±2.97)%,(73.73±1.14)%,(79.19±1.79)% and(83.88±0.94)%,respectively.The RGC apoptosis rates of control group,RGC scratch group,ET low dose group,ET medium dose group and ET high dose group were(5.08±0.17)%,(18.67±1.24)%,(17.96±0.74)%,(15.11±0.56)% and(11.67±1.32)%,respectively.Comparison of RGC survival rate between groups:compared with RGC scratch group,the cell survival rate of ET low-dose group,ET medium-dose group and ET high-dose group was increased,and the difference between RGC scratch group and ET low-dose group was not statistically significant(P=0.728);the differences between RGC scratch group,ET medium dose group and ET high dose group were statistically significant(P<0.001);the difference between ET medium dose group and ET high dose group was statistically significant(P=0.002).Comparison of apoptosis rate of RGC among groups:the apoptosis rate of RGC scratch group was significantly higher than that of control group,the difference was statistically significant(P<0.001).Compared with RGC scratch group,the apoptosis rate of ET low-dose group,ET medium-dose group and ET high-dose group was decreased,and there was no statistical significance between RGC scratch group and ET low-dose group(P=0.869).The differences of apoptosis rate between RGC scratch group,ET medium dose group and ET high dose group were statistically significant(P<0.05).The difference of apoptosis rate between ET medium dose group and ET high dose group was statistically significant(P=0.007).Conclusion ET has neuroprotective effect on RGC cultured in vitro with mechanical injury,and the protective effect increases with the increase of ET dose in a certain range.