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目的在开放式压力控制培养系统作用下体外培养大鼠视网膜神经细胞(retinal neurons ,RNs) ,观察米诺环素对其活性及凋亡的影响,并进一步探讨其对受损RNs保护的可能机制。方法采用出生0 ～3d的Sprague Dawley(SD)大鼠视网膜神经细胞体外混合培养,制备RNs加压培养模型。通过细胞形态学观察、四唑盐( MTT)比色法测定细胞活力、吖啶橙/溴化乙锭(AO/EB)染色法检测细胞的凋亡率来观察米诺环素对上述损伤细胞的保护及治疗作用,以及应用免疫细胞化学染色法,观察诱导型一氧化氮合酶(iNOS)和半胱天冬酶-3(caspase-3)表达的改变。结果加压培养后,在倒置显微镜下RNs与对照组相比形态改变较明显,细胞活力降低,大量细胞发生凋亡(占53 .93 %) ,而米诺环素治疗组(20μmol/L)细胞则形态改善,活力显著增高,凋亡数目减少(占17 .29 %) ,差异具有显著性(P<0 .05)。免疫细胞化学染色法示米诺环素治疗组细胞内iNOS和caspase-3表达较加压损伤组减少。结论一定剂量的米诺环素在体外可有效抑制压力引起的大鼠视网膜神经细胞损伤及凋亡,抑制iNOS和caspase-3的表达可能是其潜在作用机制。
Objective To investigate the effects of minocycline on the activity and apoptosis of rat retinal neurons (RNs) under the action of an open pressure-controlled culture system and to explore its possible mechanism of protecting the damaged RNs . Methods Retinal nerve cells of Sprague Dawley (SD) rats, born 0-3 days old, were cultured in vitro and cultured under pressure. Cell viability was measured by MTT colorimetric assay and apoptotic rate was detected by AO / EB staining. The effect of minocycline on the cell injury And the changes of iNOS and caspase-3 expression were observed by immunocytochemical staining. Results Under the inverted microscope, the morphological changes of RNs were obvious compared with the control group, the cell viability decreased, a large number of cells were apoptosis (53.93%), while the minocycline group (20μmol / L) The morphology of the cells was improved, the vitality was significantly increased, the number of apoptotic cells decreased (17.29%), the difference was significant (P <0.05). Immunocytochemical staining showed that the expression of iNOS and caspase-3 in the cells treated with minocycline was significantly lower than that in the untreated group. Conclusions Minocycline at a certain dose can effectively inhibit the damage of retinal nerve cells and apoptosis induced by stress in vitro, and the inhibition of iNOS and caspase-3 expression may be its potential mechanism.