点带石斑鱼脑组织和视网膜中神经型一氧化氮合成酶的分布与定位

【目的】研究点带石斑鱼脑组织和视网膜中的一氧化氮（NO）含量及神经型一氧化氮合成酶（nNOS）分布与活性，为深入揭示NO在点带石斑鱼神经系统中的作用机制打下基础。【方法】采用NADPH-d组织化学染色法、免疫组织化学法、蛋白免疫印迹（Western blotting）及Griess试剂法，对点带石斑鱼脑组织和视网膜中nNOS的分布与活性及NO含量进行分析。【结果】经NADPH-d组织化学染色后，一氧化氮合成酶（NOS）阳性物质呈蓝色。在点带石斑鱼大脑中，NOS阳性物质位于神经元和神经纤维；在小脑中，NOS分布在颗粒层的颗粒细胞、分子层的神经纤维和浦肯野细胞层的浦肯野细胞；在视网膜中，NOS分布于色素上皮层、视锥视杆层、外核层、外网层、内核层、内网层、节细胞层和视神经纤维层。经免疫组织化学染色后，nNOS阳性物质呈深棕色。在大脑中，nNOS存在于神经元和神经纤维；在小脑中，nNOS分布在颗粒层、分子层和浦肯野细胞层；在视网膜中，nNOS分布在色素上皮层、视锥视杆层、外核层、外网层、内核层、内网层、节细胞层和视神经纤维层。Western blotting检测结果显示，点带石斑鱼脑组织和视网膜中的nNOS在显色后的硝酸纤维素膜上均出现3条免疫印迹条带，对应分子量分别为80、120和130kD。NO含量在点带石斑鱼脑组织和视网膜中分别为17.601±1.743和13.624±1.249μmol/L，nNOS活性在脑组织和视网膜中分别为38.070±3.047和23.748±2.860U/mg。【结论】在点带石斑鱼脑组织和视网膜中均存在nNOS，推测nNOS在点带石斑鱼脑神经及视神经系统生理活动中发挥重要作用。脑组织中的NO含量和nNOS活性均显著高于视网膜，是由于脑组织作为重要的中枢神经，对机体生理功能起重要调节作用。

Immunohistochemical distribution and localization of neuronal nitric oxide synthase in brain and retina of Epinephelus coioides

【Objective】The content of nitric oxide（NO），distribution and activity of neuronal nitric oxide synthase （nNOS）in brain and retina of Epinephelus coioides were studied to provide foundation for further revealing the mechanism of action of NO in the nervous system of E. coioides.【Method】NADPH-d staining，immunohistochemical method， western blotting and Griess reaction were used to study the distribution，activity of nNOS and NO content in the brain and retina.【Result】The positive material of nitric oxide synthase（NOS）was stained blue using NADPH-d method. NOS positive materials were found in neurons and nerve fibers in the cerebrum. In the cerebellum，NOS was distributed in granular cells of the granular layer，nerve fibers of the molecular layer，and Purkinje cells of the Purkinje cell layer. In the retina， the NOS was found to be distributed in the pigment epithelium layer，rod and cone layer，outer nuclear layer，outer plexiform layer，inner nuclear layer，inner plexiform layer，ganglion cell layer and retinal nerve fiber layer. In addition，the immunohistochemical positive materials of nNOS were stained dark brown. In the cerebrum，nNOS was distributed in neurons and nerve fibers. In the cerebellum，nNOS was distributed in the granular layer，molecular layer and Purkinje cell layer. In the retina，nNOS was found to be distributed in the pigment epithelium layer，rod and cone layer，outer nuclear layer，outer plexiform layer，inner nuclear layer，inner plexiform layer，ganglion cell layer and retinal nerve fiber layer. The result of western blotting showed that there were three protein blotting bands on the nitrocellulose membrane after color development for nNOS in the brain and retina，their corresponding molecular weights were 80，120 and 130 kD，respectively. The NO content in brain and retina were 17.601±1.743 and 13.624±1.249 mol/L，respectively. The nNOS activity in brain and retina were 38.070±3.047 and 23.748±2.860 U/mg，respectively.【Conclusion】nNOS exists in brain and retina of E. coioides，and it is speculated that nNOS may play an important role in the cranial and visual nervous system physiological activity of fish. The NO content and nNOS activity in brain are significantly higher than those in retina，presumably because the brain is the important central nervous system and plays an important role in regulating the physiological functions of body.