| Objective: Excessive angiogenesis is a critical cause of blindness. Among them choroidal neovascularization has a large propotion. Choroidal neovascularization and its hemorrhage, exudation and proliferation are major pathologic processes in many ocular diseases. Angiogenesis results from many factors. The studies in the past are more focused on a single factor to control angiogenesis. These methods have some inhibitory effect; however, they are weak and can not invent the neova- scularization to occur or to recur. The recent studies revealed that complement activation via alternative pathway is critical in the development of laser-induced choroidal neovascularization. In order to reveal the effects of alternative pathway, in this study, we use complement factor B (CFB), an important factor in alternative pathway, to block the alternative pathway. And observe the inhibitory effects of alternative pathway on choroi- dal neovascularization.Methods: 1 Construction of CFB-siRNA expression vector1.1 Design CFB-siRNA on the basis of human's factor B mRNA. And make it homologous to rat.1.2 Construct an expression vector of CFB-siRNA by cut CFB-siRNA and plasmid pRNAT-U6.1/Neo. Translate into E.coli, optimization. Recombinant vectors were confirmed by the digestion analysis of restriction endonuclease and all inserted sequences were verified by DNA sequencing.1.3 Transfection the recombinant plasmid pRNAT-U6.1- /CFB-siRNA into human umbilical vein endothelial cells (ECV-304) by electroblot, named as CFB-siRNA group; Transfection the same quantity simple plasmid into ECV-304 cells, named as simple plasmid group; Non-transfected cells were named as normal control group.1.4 Observation the transfective efficiency with inversion fluorescence microscope 48h after transfection. And then collection the ECV-304 to RT-PCR, analysis the electrophoresis strip with Bandleader software; Detection growth inhibition ratio with MTT and growth cycle with flow cytometry.1.5 Statistical analysis: Gray value of electrophoresis strip was presented as±s; Statistical analysis was performed by t-test and an one-way ANOVA. Enumeration data were analyzed byχ2 test. P<0.05 was considered statistically significant.2 The vivo experiment of CFB-siRNA inhibits the angiogenesis of laser-induced CNV2.1 Construction Laser-induced CNV model: Experim- ental CNV was induced by laser photocoagulation. 48 Brown Norway rats with CNV were randomly divided into 7 groups, 6/group: (1)CFB-siRNA tail intravenous injection group; (2)simple plasmid tail intravenous injection group; (3)CFB- siRNA intravitreal injection group; (4)simple plasmid intravitreal injection group; (5)CFB-siRNA subretinal injection group; (6)simple plasmid subretinal injection group; (7)control group.2.2 Injection on the first, third, fifth day after photocoagulation except control group. Observation the crystal, vitreous body and retina after injection.2.3 Detection angiogenesis 7days and 14days by FFA after photocoagulation.2.4 Detection the expression of angiogenesis related factors VEGF, factorⅧby immunohistochemistry.2.5 Statistical analysis: Enumeration data were analyzed byχ2 test. Absorbance of immunostaining was presented as±s; Statistical analysis was performed by t-test and an one-way ANOVA. P<0.05 was considered statistically significant.Results: 1 Construction of CFB-siRNA expression vector1.1 We successfully constructed the CBF-siRNA vector and purified. Sequencing of the vector was identical to the sequence of design.1.2 We used ECV-304 to validate the transfective efficiency of vector. Each group except the normal control group was successfully transfected.1.3 Collection ECV-304 cell, extraction total RNA, reverse transcription to DNA, Dection the inhibitory effects of the vector by RT-PCR. Compare to simple plasmid the vector had better effect.1.4 The CFB-siRNA inhibition ratio was detected by MTT as 23.45%, 33.48%, and 45.49% respectively at 24h, 48h and 72h. The simple plasmid had no influence on ECV-304.1.5 Flow cytometry showed that CFB-siRNA inhibited the ECV-304 at the G1 intermediate stage of the growth cycle.2 The vivo experiment of CFB-siRNA inhibits the angiogenesis of laser-induced CNV2.1 Experimental CNV was induced by laser photocoagulation. 48 Brown Norway rats with CNV were randomly divided into 7 groups, 6/group: (1)CFB-siRNA tail intravenous injection group; (2)simple plasmid tail intravenous injection group; (3)CFB-siRNA intravitreal injection group; (4)simple plasmid intravitreal injection group; (5)CFB-siRNA subretinal injection group; (6)simple plasmid subretinal injection group; (7)control group.2.2 On the 1d, 2d, 3d after vitreous injection and subretinal injection, the crystal, vitreous and retina were transparent, withthout inflammatory reaction.2.3 After injection, fluorescein leakage of siRNA injection groups from the CNV lesions was significantly decreased compared with the control eyes; Compared with vitreous injection group and subretinal injection group, fluorescein leakage of intravenous injection group from the CNV lesions decreased significantly(P<0.05). 2.4 VEGF, factorⅧimmunostaining was similar between 7d and 14d after laser in CFB-siRNA in tail intravenous injection group(P>0.05). While compared with the other groups on the same time, the immunostaining was weak obvious (P<0.05). And VEGF, factorⅧimmunostaining of 14d after laser was stronger than that of 7d after laser for the other groups (P<0.05).Conclusion: 1. Construction of CFB-siRNA inhibited the expression of complement factor B effectively, providing the tool for the second stage.2. Construction of CFB-siRNA was transfected into ECV-304 by electroblot successfully, and inhibited the proliferation of ECV-304.3 CNV model was induced by laser successfully, which method was easy, less time and high successful rate.4 Construction of CFB-siRNA inhibited the laser-induced CNV and down-regulated the expression of VEGF and factorⅧ.5 The result showed that tail intravenous injection was the best method.6 All these results indicated that inhibition of the factor B expression may be useful in the gene therapy for the CNV. |
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