RNA interference retinitis pigmentosa animal studies
|School||Second Military Medical University|
|Keywords||Retinitis pigmentosa RNA interference siRNA Mice Rhodopsin|
Purpose 1. This experiment gene targeting construct carrying mutant human RHO gene retinal degeneration in mice. For building a successful rat-old mice of different pathological changes of retina, to clarify its pathological features. (2) Preparation of gene targeting RHO LV-GFP-shRNA. By microscopic syringe lentiviral vector injected into the cavity under the mouse retina. Studied at different times after injection hRHO mRNA and protein expression; different times retina and retinal transfection efficiency morphological changes. RNA interference technique to assess the therapeutic effect, provide experimental basis for clinical application. Method 1. Construct mRHOp-hRHO-SV40A-PBS plasmid injection in mice zygotes, bred transgenic mice. RT-PCR and western-blot method for identification of transgenic mice. 2.18 RP mice were divided into 4 groups. In mice 15 days after birth, 30 days, 60 days were sacrificed 6. 5 healthy mice were compared. Intraperitoneal injection of sodium pentobarbital overdose mice were killed immediately after the complete removal of the mouse eye, along the optic nerve to do 3mm thick slices. HE staining, in situ detection of apoptosis and electron microscopy AMD testing. Compare different time retinal outer nuclear layer thickness variations, the positive rate of photoreceptor cell apoptosis and ultrastructural changes of photoreceptor cells. Results using T test between groups, spss13.0 statistical software for statistical analysis. 3 Use the pre-vitro transfection experiments confirmed the efficiency of gene silencing was 79.14% of the siRNA sequences targeting hRHO construct pGCL-GFP-shRNA lentiviral shuttle plasmid and prepare LV-GFP-shRNA. 4.80 RP mice rats aged 15 days, the left eye as a control group without any treatment; eye subretinal injection of LV-GFP-shRNA. 15 days after injection, respectively, 30 days, 45 days, 60 days random mice were sacrificed 20 RP. Semi-quantitative RT-PCR and western-blot was used to detect various times after treatment, RP mouse retina hRHO mRNA and protein expression. Retinal stretched observed at different times after treatment of retinal transfection efficiency, retinal pigment cells staining with a range of 0-3 assessments. (0: no fluorescent cells found; 1: scattered positive cells; 2: some positive cells together; 3: Continuous positive cells). OK immunofluorescence staining RHO retina expressed by HE staining outer nuclear layer thickness. Results 1. MRHOp-hRHO-SV40-PBS plasmid correct position by a variety of restriction endonuclease identified after sequencing. Linearized plasmid locus for the NotI and Pvul, double digestion can be cut into 3037bp (recycling) and 1679bp, 1043bp, 3037bp for recycling microinjection. After injection of mouse zygotes successfully bred mutant gene expression hRHO RP mice. 2.RP mice 15 days after birth, retinal outer nuclear layer and the core layer can already separate the layers. The normal control group, the outer nuclear layer of the retina at each time point did not change significantly (9.30 ± 0.37), and mice were born 15 days RP ONL thinning starts (7.05 ± 0.25), 30 天 significantly thinner (5.29 ± 0.24), 60 days reduced outer nuclear layer monolayer (2.04 ± 0.28). TUNEL staining was negative control group, RP mice 15 days had scattered positive granules, 60 days there was a large positive particles. Photoreceptor cells prompted massive necrosis and apoptosis. Transmission electron microscopy revealed: Early retinal rod outer segment normal external membrane continuous, 30 days begin to shorten the rod outer segment, external membrane begins to thin, not continuous. Disk membrane vacuoles like change, inner segment mitochondria, endoplasmic reticulum variability in varying degrees, dissolve; nuclei appeared nuclear enrichment, the occurrence of apoptosis. 3 targeting hRHO successfully constructed the pGCL-GFP-shRNA 'lentiviral shuttle plasmid and prepared a titer of 1.0 × 1010ifu/ml the LV-GFP-shRNA. 4.RP mice carrying the right eye LV-GFP-shRNA 30 days after injection of RHO mRNA expression began to decline, to 60 days decreased significantly. Group comparison: in 45 days, 60 days mRNA expression LV-GFP-shRNA group compared with the negative control group decreased significantly (P lt; 0.05). At the protein expression level, LV-GFP-shRNA group compared with the control group was 30 days began to decline, further decline in 45 days, 60 days time and stabilized. Group comparison: in 45 days, 60 days protein levels LV-GFP-shRNA group compared with the negative control group decreased significantly (P lt; 0.05). ONL retinal layers change LV-GFP-shRNA group than in the negative control group at 15 days, 30 days, 45 days were not significantly different from 60 days to appear LV-GFP-shRNA group than in the negative control group ONL layer thickening (P lt; 0.05). Conclusions 1. Use of transgenic technology successfully constructed mutant gene expression hRHO retinal degeneration in mice. Provide a reliable treatment for further study in animal models. (2) The experimental mouse model constructed by RP histopathological changes in the retina of the human RHO gene mutations induced retinitis pigmentosa pathological changes similar for the next row determine the efficacy of gene therapy provide the morphological basis. 3 targeting hRHO successfully constructed the pGCL-GFP-shRNA lentiviral shuttle plasmid and prepared a titer of 1.0 × 1010ifu/ml the LV-GFP-shRNA. 4. LV-GFP-shRNA shortly after subretinal injection of RHO proteins can be significantly reduced. RNA interference technology for the treatment of human retinitis pigmentosa provide a reliable basis for animal experiments.