摘要
背景缺血缺氧损伤是引起大部分视觉系统损害的主要原因，目前尚无有效药物从根本上缓解缺血缺氧带来的损害。研究发现，微小RNA（miR）-30b有助于减轻缺血缺氧导致的心肌损伤，但其对糖氧剥夺的视网膜神经节细胞（RGCs）生存是否有类似的保护作用鲜有文献报道。目的研究重组腺相关病毒介导的miR-30b转染对糖氧剥夺RGCs的保护作用。方法取出生后24h的8只sD大鼠眼球，剥离出视网膜组织以进行RGCs的原代培养，将原代培养的细胞分为重组腺相关病毒（rAAV）-miR对照组、rAAV—miR．30b拟似物组、rAAV—miR-30b抑制物组和PBS组，分别在培养液中添加rAAV—miRNA、rAAV—miR-30b拟似物、rAAV—miR-30b抑制物或PBS培养细胞6d，RGCs：AAV为1：10000。各组细胞分别进行低氧培养箱（37℃，体积分数5％CO2、17％N2、3％O2）联合低糖（葡萄糖质量浓度为1．0g／L）培养液进行培养以建立原代糖氧剥夺RGCs模型，并与正常培养（37℃、5％CO2）的细胞进行对照。采用细胞计数试剂盒-8（CCK-8）法检测各组细胞活力，采用免疫荧光染色法检测各组细胞中神经元特异性标志物TubulinⅢ的表达，并计算存活RGCs数目。采用Hoechst／PI染色法检测各组细胞的凋亡和坏死情况。结果培养7d的正常成熟RGCs可见1—3条完整的细长神经元突起及其分支。糖氧剥夺后随着时间延长，培养的RGCs逐渐减少和破坏，突起的主干结构破碎。rAAV·miR-30b拟似物组细胞相对活性分别为3．310±0．162，明显高于rAAV—miR-30b抑制物组和rAAV—miR对照组的0．949±0．141和0．900±0．181，差异均有统计学意义（t=10．508、10．296，均P〈0．001）。存活的RGCs可表达Tubulinm，呈红色荧光。rAAV—miR-30b拟似物组TubulinlI阳性细胞数量为（13．800±1．924）／视野，明显多于rAAV—miR-30b抑制物组的（0．600±0．548）／视野和rAAV—miR对照组的（0．800±1．304）／视野，差异均有统计学意义（t=15．141、14．912，均P〈0．001）。rAAV—miR-30b拟似物组、rAAV—miR对照组和PBS组细胞凋亡率和死亡率的总体比较差异均有统计学意义（F=10．851，P=0．002；F=6．378，P=0．013），rAAV—miR-30b拟似物组细胞凋亡率和死亡率均明显低于rAAV—miR对照组和PBS组，差异均有统计学意义（均P〈0．05）。结论低氧培养箱联合低糖培养液建立稳定的原代RGCs糖氧剥夺模型；rAAV介导的miR-30b转染可抵抗糖氧剥夺对RGCs的损伤。
Background Retinal ganglion cell （RGCs） death following ischaemic insuh is the major cause of a number of vision-threatening diseases. Recent studies confirmed that micro RNA （miR-30b） can alleviate hypoxy-induced cardiac injury. However, whether miR-30b can protect RGCs against oxygen-glucose deprivation damage is still not ellucidated. Objective The aim of this study was to investigate the protective effect of miR-30b on RGCs damage caused by oxygen-glucose deprivation. Methods The retinas were isolated from the eyeballs of eight SD rats aged postnatal 24 hours and RGCs were primarily cultured. The ceils were divided into the recombinant adeno-associated virus （rAVV） control group,rAAV-miR-30b mimic group and AAV-miR-30b inhibitor group. Then the cells were transfected using rAVV-miR plasmid, rAAV-miR-30b mimic plasmid and AAV-miR-30b inhibitorplasmid,respectively for 6 days with the RGCs： AAV as 1 ： 10 000. The cells were cultured with low glucose medium in hypoxygen incubator （5% CO2,17% N2 ,3% O2 ） or 5% CO2 incubator respectively for 24 hours. Cell viability was detected by cell counting kit-8 assay. The expression of Tubulin Ⅲ, a neuron specific marker, was detected by immunofluorescence technology to evaluate the survival of RGCs. The apoptosis and necrosis of the cells were assessed by Hoechst/PI double staining. Results The RGCs grew well with round shape and 1-3 processes 7 days after cultured in the normal cells. However,the RGCs were diminished and the cell process disrupted in the oxygen-glucose deprivation group. The relative vability of the cells was 3. 310 ±0. 162 in the rAAV-miR-30b mimic group,which was significantly higher than 0. 949 ±0. 141 in the rAAV-miR-30b inhibitor group and 0. 900 ±0. 181 in the rAAV-miR control group（t= 10. 508,10.296,both at P〈0.001 ）. It was positively expressed in survival RGCs, with the red fluorescence. The number of Tubulin cells was （13. 800± 1. 924）/field in the rAAV-miR-30b mimic group, showing a significant increase in comparison with （0. 600±0. 548）/field in the rAAV-miR-30b inhibitor group and （0. 800± 1. 304 ）/field in the rAAV-miR control group （t = 15. 141, 14. 912, both at P 〈 0. 001 ）. Significant differences were found in the apoptosis rate and necrosis rate among the rAAV-miR-30b mimic group, rAAV-miR control group and PBS group （F = 10. 851, P = 0. 002;F = 6. 378 ,P = 0. 013 ） , and the apoptosis rate and necrosis rate in the rAAV-miR-30b mimic group were considerably lower than those in the rAAV-miR control group and PBS group （ all at P〈0.05）. Conclusions The oxygen-glucose deprivation models can be established in RGCs by hypooxygic and low-glucose cultivation, rAAV encoding miR-30b mimics transfection can protect RGCs against oxygen-glucose deprivation damage.
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2016年第5期396-401,共6页
Chinese Journal Of Experimental Ophthalmology
基金
国家自然科学基金项目（81371006、81300762）
