人源Pro370Leu突变Myocilin诱导转基因小鼠开角型青光眼表型的研究

Abstract

To investigate the characteristics of mutation myocilin proteins and glaucoma pathological phenotype in transgenic mice with full-length human Pro370Leu mutant myocilin gene (Tg- MYOC P370L ). Tg- MYOC P370L mice were established using the CRISPR/Cas9 system. Long-term intraocular pressure (IOP) was measured, myocilin protein expressions in anterior chamber angle, retina, optic nerve tissues and aqueous humor were detected by western blot. RBPMS, myocilin, Iba-1 and GFAP expression were visualized by immunofluorescence. H&E staining was applied to assess the ocular angle and retinal morphology. Aqueous humor dynamics were visualized by Gadolinium magnetic resonance imaging (Gd-MRI). TUNEL assay was used to evaluate the specific cell apoptosis in trabecular meshwork and retina. Optomotor and electroretinography tests were employed to evaluate the visual function in Tg- MYOC P370L and wild-type (WT) mice. Homozygous myocilin mutation at position 503 (C > T) was identified by PCR and sequencing in Tg- MYOC P370L mice. Myocilin protein expression was overexpressed in eye tissues of Tg- MYOC P370L mice with reduced myocilin secretion in aqueous humor. H&E staining showed normal histological morphology of anterior chamber angle whereas decreased thickness and nuclei in ganglion cell layer were found ( P  < 0.05). Gd signals were significantly increased in the anterior chamber of Tg- MYOC P370L compared with WT eyes ( P  < 0.05). IOP was elevated in Tg- MYOC P370L mice starting at 5 months of age, with significant RGC loss ( P  < 0.05). Upregulation of caspase-3 and caspase-9 expressions and increased TUNEL-positive cells were found in eyes of Tg- MYOC P370L mice. Excessive activation of retinal glial cells and impaired visual function were detected in Tg- MYOC P370L mice. Tg- MYOC P370L mice can induce the phenotype of open-angle glaucoma, featured as IOP elevation, activated retinal glial cells, loss of RGCs and impaired visual function. These pathologic change