[Zhonghua yan ke za zhi] Chinese journal of ophthalmology · 2010
[VEGF short hairpin RNA expression plasmid blocks VEGF expression in rat vascular endothelial cell and inhibits rat corneal neovascularization].
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Fei Chun-mei, Zhou Shan-bi
Аннотация
OBJECTIVE: To investigate RNA interference in rat corneal neovascularization and vascular endothelial cells of vascular endothelial growth factor expression and its mechanism.
METHODS: In empirical study short hairpin RNA (shRNA) targeting VEGF mRNA was designed and synthesized and VEGF shRNA eukaryotic plasmids were transfected into EC with Lipofectamine 2000 transfection system. Expression of VEGF mRNA and protein in transfected cells was detected by RT-PCR and Western blot. Twenty SD rats were randomized into two groups by randomly Design, experimental group were 10 and 10 in the control group. CNV was induced in vivo by alkaline cauterization of the central cornea, the rats received subconjunctival injection of VEGF shRNA3 plasmid. Then corneal neovascularization was evaluated with microscope. After seven days, VEGF mRNA and protein was determined by RT-PCR and Western blot respectively.
RESULTS: VEGF shRNA expression plasmids were successfully constructed and identified by restriction enzyme and sequence analysis. VEGF expression was down regulated by VEGF shRNA1, 2, 3 plasmids, and VEGF shRNA3 plasmid showed stronger inhibitory effect. The inhibitory effects of VEGF mRNA and protein are 68.92% and 66.22%. At the 3, 5 and 7 days after alkali cauterization, the CNV length of the experimental group was obviously decreased comparing with the control group, the difference between two groups has statistics meaning (F=402.700, P=0.000); the CNV area of the experimental group was also obviously decreased comparing with the control group, the difference between two groups has statistics meaning (F=402.700, P=0.000); the VEGF mRNA and protein in corneas was also decreased, the inhibitory effects of which are 41.84% and 41.86%.
CONCLUSIONS: RNAi significantly suppresses expression of VEGF in rat EC in vitro. In vivo, subconjunctival delivery of VEGF shRNA3 plasmid suppresses injury-induced VEGF expression and CNV.
METHODS: In empirical study short hairpin RNA (shRNA) targeting VEGF mRNA was designed and synthesized and VEGF shRNA eukaryotic plasmids were transfected into EC with Lipofectamine 2000 transfection system. Expression of VEGF mRNA and protein in transfected cells was detected by RT-PCR and Western blot. Twenty SD rats were randomized into two groups by randomly Design, experimental group were 10 and 10 in the control group. CNV was induced in vivo by alkaline cauterization of the central cornea, the rats received subconjunctival injection of VEGF shRNA3 plasmid. Then corneal neovascularization was evaluated with microscope. After seven days, VEGF mRNA and protein was determined by RT-PCR and Western blot respectively.
RESULTS: VEGF shRNA expression plasmids were successfully constructed and identified by restriction enzyme and sequence analysis. VEGF expression was down regulated by VEGF shRNA1, 2, 3 plasmids, and VEGF shRNA3 plasmid showed stronger inhibitory effect. The inhibitory effects of VEGF mRNA and protein are 68.92% and 66.22%. At the 3, 5 and 7 days after alkali cauterization, the CNV length of the experimental group was obviously decreased comparing with the control group, the difference between two groups has statistics meaning (F=402.700, P=0.000); the CNV area of the experimental group was also obviously decreased comparing with the control group, the difference between two groups has statistics meaning (F=402.700, P=0.000); the VEGF mRNA and protein in corneas was also decreased, the inhibitory effects of which are 41.84% and 41.86%.
CONCLUSIONS: RNAi significantly suppresses expression of VEGF in rat EC in vitro. In vivo, subconjunctival delivery of VEGF shRNA3 plasmid suppresses injury-induced VEGF expression and CNV.