压力对大鼠纯化视网膜神经节细胞诱导型一氧化氮合酶mRNA及其蛋白表达的影响

Pressure influence on mRNA and protein expression of inducible nitric oxide synthase in purified retinal ganglion cells of rats

目的 研究不同压力下纯化培养的大鼠视网膜神经节细胞 (retinalganglioncells ,RGCs)中诱导型一氧化氮合酶 (induciblenitricoxidesynthase ,iNOS)mRNA及其蛋白质的表达 ,探讨青光眼患者RGCs损伤的机制。方法 将纯化培养的Sprague Dawley大鼠RGCs随机分成对照组和A、B、C、及D组 ,分别在 0、2 0、4 0、6 0、及 80mmHg(1mmHg =0 133KPa)的压力下培养 4 8h后 ,用原位杂交、RT PCR及Westernblot法检测RGCs中iNOSmRNA及其蛋白质的表达 ,并用全自动图像分析系统测定其吸光度值。结果 纯化培养的RGCs纯度为 98%。原位杂交、RT PCR及Westernblot法检测RGCs中iNOSmRNA及其蛋白表达结果变化均呈平行关系 :对照组无表达 ,A组有较弱的表达信号 ,B、C及D组表达逐渐增强 ;3项检测结果用全自动图像分析显示 :A组iNOSmRNA均弱表达 ,与对照组比较差异有显著意义 (P <0 0 5 ) ;B、C及D组均为强表达 ,各组与对照组比较差异有非常显著意义(P <0 0 1)。结论 压力可激活RGCs中iNOSmRNA及其蛋白质的表达 ,由此产生过量的NO损伤RGCs,成为青光眼的发病因素之一。

Objective To study the inducible nitric oxide synthase (iNOS) mRNA and protein expression in rat purified retinal ganglion cells (RGCs) that cultured under different pressures. Methods (1) To culture RGCs that from Sprague Dawley (SD) neonatal rats (postnatal 1-5 days) on two planes in assimilative culture solution, RGCs were purified by Thy1.1 with sheep anti rat FITC monoclonal antibody. (2) RGCs were cultured under pressures of 0, 20, 40, 60 and 80 mm Hg, respectively. The changes of iNOS mRNA and protein in RGCs under different pressures were demonstrated qualitatively and quantitatively by in situ hybridization, RT PCR and Western blot. Results After cultured for 12 and 24 hours in vitro, the purification rate of RGCs in the experiment reached 98%. The expression of iNOS mRNA and protein in RGCs became higher and higher as the pressure was increased. There were no iNOS mRNA and protein expression in the control group (0 mm Hg) and weak expression in 20 mm Hg group ( P <0.05). 40, 60 and 80 mm Hg groups had a very significant difference from the control group, respectively ( P <0.01). Conclusion Pressure can evoke the expression of nitric oxide synthase mRNA and protein in purified retinal ganglion cells in vitro, thus the nitric oxide can be one of the causes of RGC damage, that may induce or aggravate glaucoma.