Inhibition Efficacy Of CFB-siRNA In Laser-induced Choroidal Neovascularization In Rats

Objective:Coroidal neovascularizationh (CNV) is an important ocular manifestation of angiogenesis in eyes, which derives from the choroid capillaries, these vessels grow and make their way through Bruch membrane and orient themselves in the plate between the Bruch membrane and RPE layer( retinal pigment epithelium) or between RPE layer and the retinal sensory layer . CNV is one of the leading factors resulting in vision loss in the developing courses of various fundus diseases. Current clinical therapies for CNV include laser photocoagulation, photodynamic therapy (PDT), transpupillary thermotherapy (TTT), operation divestment, radiotherapy, etc, however, none of these therapies produces completely satisfactory clinical outcomes, many problems are still remained such as high recurrence rate, the inevitability of normal tissue damage, further aggravation of visual function. Recent studies have found that complement activation and membrance attrack complex (MAC) deposition are playing a key role in the laser-induced CNV .In the current study, we utilized a new gene silencing technology---RNA interference (RNA interference, RNAi), to mutate CFB and blocked this complement activation pathway; through this approach we study the role of CFB in CNV.Methods:1 The evaluation of transfection efficiency by differernt concentrations of CFB-siRNA1.1 The 60 rats of 8-10 weeks healthy Brown Norway(BN),were randomly divided into four groups of fifteen. Krypton laser (wavelength 647nm,spot diameter of 200μm,power of 260mW,exposure time 0.05s) around the optic disc photocoagulation 9-10 points,see any air bubbles produced by the breakdown Bruch membrane. 1.2 The 60 BN rats,were randomly divided into control group, low dose group(25μgCFB-siRNA),mid dose group(50μgCFB-siRNA) and high dose group(75μgCFB-siRNA) .Control group and three test group are treated with laser photocoagulation for CNV rat model.Test group is injected through the tail vein .Injection Time: The day before the peak of CFB expression; Injection: Tail vein injection, injected once every other day,a total of three injections. The observation time is 3d,7d,14d,21d,28d after photocoagulation.1.3 Fundus fluorescein angiography (FFA) was performed on days 3,7, 14, 21,28.1.4 6h after angiography, the rats were killed,removal of posterior segment, make paraffin sections, select a clear laser spot, HE staining.1.5 Randomly selected the paraffin sections on 3,7,14,21,28 day after photocoagulation,make immunohistochemical staining.1.6 Image analysis and statistical analysis By SPSS16.0 software,the data of gray value at each time point is analyzed, observed the expression of VEGF,FactorⅧ.2 Observed the relation beteen CFB and the CNV-related VEGF,TGF-β2 in rats after photocoagulation.2.1 Randomly selected the sections from the control group and high dose group (75μgCFB-siRNA), part of the tissue samples from the first part.2.2 Observed the expression of CFB,VEGF,and TGF-β2 in each group though immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR)on days 7, 14, 21,28.2.3 The data were statistically analyzed by SPSS16.0.3 Toxicity studies of CFB-siRNA on the retina.3.1 Randomly selected the sections from the control group and high dose group (75μgCFB-siRNA), part of the tissue samples from the first part. and added the control group.3.2 Rats were sacrificed respectively at 7,14,21,28 d, removal of posterior segment eye tissues , making paraffin sections. Selected the tissue biopsy far from the laser spot , routine HE staining and electron microscopy. Results:1 The evaluation of transfection efficiency by differernt concentrations of CFB-siRNA1.1 CNV was found on 7d after photocoagulation, 14d gradually increased, and reached the peak on 21d.1.2 FFA showed hyperfluorescence in early stage and fluorescence leakage significant in late stage.1.3 The incidence of CNV reached a peak on 21d in test group after photocoagulation , but was lower than the control groupsignificantly.It shows the test group was significantly lower than control group. And the high-dose treatment group was significantly lower than the low-dose treatment group compared by FFA. (P <0.05).1.4 It was identified that CNV invade subretinal space or under RPE by light and electron microscope.Aslo macrophages,RPE cells,fibroblasts were seen.1.5 Immunohistochemical staining showed that VEGF, FactorⅧexpression decreased than the control group, and the decrease level of high-dose treatment group was higher than the low-dose group2 Observed the relation beteen CFB and the CNV-related VEGF,TGF-β2 in rats after photocoagulation.2.1 The immunohistochemistry showed that CFB appeared before VEGF and TGF-β2 in control group ,it decreased after 7 and was still a small amount of expression after 14 d. VEGF and TGF-β2 increased significantly in the 14-21d after photocoagulation, then decreased slightly. Compared with the control group, the expression of CFB reduced in 7d, then approached to the control group gradually, the expression of VEGF and TGF-β2 were significantly reduced in the experimental treatment group.2.2 RT-PCR results showed that the expression of CFB continued to increase, VEGF , and TGF-β2 showed a curve , reached a peak expression in 21d in the control group ; In the experimental treatment group, the expression of CFB,VEGFand TGF-β2 was significantly reduced, and had no significant change at each time points. 3 Toxicity studies of CFB-siRNA on the retina.The retinal layers away from the laser spot were observed after HE staining , The structure was clear, arrange the rule, and had no obvious abnormality. The transmission electric mirror inspection result showed the structure of rod photoreceptors and cone photoreceptors were complete , but the structure of membranous disc was mild disorders, and the interval was slightly irregular. The ganglion cell layer and nerve fiber layer were edema slight , mitochondrial structure was clear , endoplasmic reticulum distended slightlyConclusion:1 Tail vein injection of CFB-SiRNA can effectively inhibit the generation of laser-induced CNV , the inhibitory effect is more apparent with the increase of the dose.2 Alternative pathway of complement play an important role in the production of CNV. It can be reduced the expression of VEGF and TGF-β2 by inhibiting the CFB.3 Tail vein injection of CFB-SiRNA have no toxicity effects on eye。...