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24 h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∶10 000.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1.0 g/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TubulinⅢ,呈红色荧光.rAAV-miR-30b拟似物组TubulinⅢ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(￡=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.

abstractsBackground Retinal ganglion cell (RGCs) death following ischaemic insult 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 cells 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 inhibitor plasmid,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 Ⅲ+ 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.