摘要背景 已有研究证实与羊毛甾醇结构相似的三萜类化合物对于全身多种疾病具有治疗作用,近年来发现羊毛甾醇合酶(LSS)及羊毛甾醇对白内障具有治疗作用,但羊毛甾醇及其抑制剂与其他眼病的关系尚不清楚.了解羊毛甾醇在眼组织中的分布有助于阐明其与眼病的关系. 目的 研究正常大鼠角膜、晶状体和视网膜中LSS及羊毛甾醇的表达及分布,为相关眼科疾病的靶向治疗提供依据.方法 取SPF级雄性SD大鼠15只,采用戊巴比妥钠过量麻醉法处死实验大鼠,立即摘取眼球,分别采用Western blot和逆转录(RT)-PCR法检测大鼠角膜、晶状体和视网膜组织中LSS蛋白及其mRNA的表达情况;采用免疫荧光化学法对LSS在角膜、晶状体和视网膜组织中的表达进行定位;采用液相色谱-质谱联用仪(LC-MS)检测羊毛甾醇在正常大鼠角膜、晶状体和视网膜组织中的含量. 结果 Western blot法检测显示,大鼠视网膜中无LSS蛋白表达,LSS蛋白在晶状体组织的相对表达量为0.43±0.05,明显高于角膜组织中的0.25±0.03,差异有统计学意义(t =-5.35,P＜0.01).RT-PCR结果显示,正常大鼠视网膜中无LSS mRNA表达,大鼠角膜和晶状体组织中LSS mRNA的相对表达量为0.51±0.04,明显高于角膜组织中的0.29±0.02,差异有统计学意义(t=-8.34,P＜0.01).免疫荧光化学法结果表明,LSS主要表达于角膜上皮、基质和内皮层角膜细胞的细胞质以及晶状体上皮细胞和浅皮质层细胞,而视网膜组织中未检测到LSS表达,且视网膜中LSS与NeuN标记的神经元及谷氨酰胺合成酶(GS)标记的Müller细胞无共表达.LC-MS检测显示,正常大鼠视网膜中未检测到羊毛甾醇,大鼠晶状体中含羊毛甾醇为(24.37±2.91) ng/mg,明显高于角膜组织中的(5.31±0.58) ng/mg,差异有统计学意义(t=-11.13,P＜0.01).结论 LSS及羊毛甾醇分布于大鼠角膜各层组织以及晶状体组织中,在视网膜各层组织中均无LSS表达.

abstractsBackground Researches showed that triterpenoids,with a similar structure to lanosterol,has therapeutical effect on many systemic diseases,and lanosterol was determined to have a therapeutical effect on cataract recently.However,how the lanosterol plays effects on other eye diseases is still unelucidated.Understanding the distribution of lanosterol in ocular tissue is helpful for us to elucidate the relationship of lanosterol with eye diseases.Objective This study attempted to investigate the distribution of lanosterol synthase (LSS) and lanosterol in cornea,lens and retina tissue of rats and offer a basis for the targeting treatment of eye diseases.Methods Fifteen SPF male SD rats were sacrificed by excessive anesthesia to obtain the eyeballs.The relative expressions of LSS protein and gene in the cornea,lens and retina tissue of the rats were detected by Western blot and reverse transcription (RT)-PCR,respectively.Immunofluorescence staining technology was used to locate the distribution of LSS in cornea,lens and retina tissue.The contents of lanosterol in the cornea,lens and retina tissue were analyzed by liquid chromatograph mass spectrometer (LC-MS).Results No LSS protein and mRNA was expressed in the retinal tissue in normal rats.The mean relative expression of LSS protein in the lens and cornea was 0.43±0.05 and 0.25±0.03,respectively,showing a significant difference between them (t =-5.35,P＜ 0.01).The relative expression of LSS mRNA was 0.51 ±0.04 and 0.29 ±0.02 in the lens and cornea,respectively,with a stronger expression in the lens in comparison with the cornea (t =-8.34,P＜0.01).Immunofluorescence staining showed that LSS primarily located in corneal epithelial layer,stromal layer and endothelial layer as well as lens epithelial cells and shallow cortex layer and hardly expressed in retina,and no co-expression of LSS with the neuron marked by NeuN and the Müller cell marked by glutamine synthetase (GS) in retinal tissue.LC-MS analysis revealed that the contents of lanosterol in lens and cornea was (24.37 ±2.91) ng/mg and (5.31 ±0.58) ng/mg,respectively,with a significant difference between them (t =-11.13,P＜0.01).Conclusions LSS and lanosterol extensively distribute in cornea and lens of normal rats,but not in retina tissue.These results offer new strategies for the target treatment of relevant eye diseases.