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Silencing of Rac1 expression via RNA interference inhibits retinal neovascularization in rats.

Li J, Li Y, Zhang M, Hu Z - Mol. Vis. (2012)

Bottom Line: Two weeks after transfection, the neonatal vessels were tested using fluorescein isothiocyanate-dextran retinal angiography.The number of endothelial cells beyond the internal limiting membrane was counted with hematoxylin and eosin staining.In the shRNA interference group, the mean number of endothelial cells beyond the internal limiting membrane was significantly higher than that in the positive control group or the interference negative control group (p<0.05).

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Second People’s Hospital of Yunnan Province, Kunming, China. juanjuanlicn@yeah.net

ABSTRACT

Purpose: To investigate the inhibitory effect of Ras-related C3 botulinum toxin substrate 1-small interfering RNA (Rac1-siRNA) on retinal neovascularization in a rat model.

Methods: Rac1-short hairpin RNA (shRNA) was synthesized, constructed, and transfected into HeLa cells. Reverse transcription polymerase chain reaction was then conducted to test for Rac1 gene expression. Retinal vein obstruction was performed in 25 Sprague-Dawley rats using the retinal photodynamic method. The vitrea bulbus of the eye in the shRNA rats group was transfected with the Rac1-shRNA vector, the other eye in the blank control group was transfected with the blank vector, and the interference control group was prepared by transfecting the Rac1-shRNA vector. Two weeks after transfection, the neonatal vessels were tested using fluorescein isothiocyanate-dextran retinal angiography. The number of endothelial cells beyond the internal limiting membrane was counted with hematoxylin and eosin staining.

Results: A large area of neovascularization and fluorescein isothiocyanate leakage was found in the positive control group. However, a small area of neovascularization and a little fluorescence leakage were found in the shRNA group, whereas the retinal vessels were normal in the negative control interference group. In the shRNA interference group, the mean number of endothelial cells beyond the internal limiting membrane was significantly higher than that in the positive control group or the interference negative control group (p<0.05).

Conclusions: Silencing Rac1 expression with RNA interference inhibits retinal neovascularization in rats.

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Related in: MedlinePlus

Cell transfection detected by fluorescence microscope. A: The image of the frozen section of rat eyeball wall in the control group under a microscope. Moderate green fluorescence was observed in the retinal region close to the vitrea bulbus 4 days after blank vector transfection. B: The image under a fluorescent microscope. Green fluorescence was seen in the retinal region. C: The image of the frozen section of rat eyeball wall in the gene interference group under a microscope. Moderate green fluorescence was seen in the retinal region close to the vitrea bulbus 4 days after recombinant vector transfection. D: The image under a fluorescent microscope. Green fluorescence was seen in the retinal region.
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f1: Cell transfection detected by fluorescence microscope. A: The image of the frozen section of rat eyeball wall in the control group under a microscope. Moderate green fluorescence was observed in the retinal region close to the vitrea bulbus 4 days after blank vector transfection. B: The image under a fluorescent microscope. Green fluorescence was seen in the retinal region. C: The image of the frozen section of rat eyeball wall in the gene interference group under a microscope. Moderate green fluorescence was seen in the retinal region close to the vitrea bulbus 4 days after recombinant vector transfection. D: The image under a fluorescent microscope. Green fluorescence was seen in the retinal region.

Mentions: The selected recombinant shRNA vector and blank control vector was transfected into the rat’s vitreous cavity successfully, and moderate intensity fluorescence was observed (Figure 1). The constructed recombinant vector and the blank vector contained the cytomegalovirus (CMV) promoter that induced the expression of downstream green fluorescent protein (GFP) genes. The fluorescence proved the recombinant vector and the blank vector effectively induced gene expression in rat retinas.


Silencing of Rac1 expression via RNA interference inhibits retinal neovascularization in rats.

Li J, Li Y, Zhang M, Hu Z - Mol. Vis. (2012)

Cell transfection detected by fluorescence microscope. A: The image of the frozen section of rat eyeball wall in the control group under a microscope. Moderate green fluorescence was observed in the retinal region close to the vitrea bulbus 4 days after blank vector transfection. B: The image under a fluorescent microscope. Green fluorescence was seen in the retinal region. C: The image of the frozen section of rat eyeball wall in the gene interference group under a microscope. Moderate green fluorescence was seen in the retinal region close to the vitrea bulbus 4 days after recombinant vector transfection. D: The image under a fluorescent microscope. Green fluorescence was seen in the retinal region.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369893&req=5

f1: Cell transfection detected by fluorescence microscope. A: The image of the frozen section of rat eyeball wall in the control group under a microscope. Moderate green fluorescence was observed in the retinal region close to the vitrea bulbus 4 days after blank vector transfection. B: The image under a fluorescent microscope. Green fluorescence was seen in the retinal region. C: The image of the frozen section of rat eyeball wall in the gene interference group under a microscope. Moderate green fluorescence was seen in the retinal region close to the vitrea bulbus 4 days after recombinant vector transfection. D: The image under a fluorescent microscope. Green fluorescence was seen in the retinal region.
Mentions: The selected recombinant shRNA vector and blank control vector was transfected into the rat’s vitreous cavity successfully, and moderate intensity fluorescence was observed (Figure 1). The constructed recombinant vector and the blank vector contained the cytomegalovirus (CMV) promoter that induced the expression of downstream green fluorescent protein (GFP) genes. The fluorescence proved the recombinant vector and the blank vector effectively induced gene expression in rat retinas.

Bottom Line: Two weeks after transfection, the neonatal vessels were tested using fluorescein isothiocyanate-dextran retinal angiography.The number of endothelial cells beyond the internal limiting membrane was counted with hematoxylin and eosin staining.In the shRNA interference group, the mean number of endothelial cells beyond the internal limiting membrane was significantly higher than that in the positive control group or the interference negative control group (p<0.05).

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Second People’s Hospital of Yunnan Province, Kunming, China. juanjuanlicn@yeah.net

ABSTRACT

Purpose: To investigate the inhibitory effect of Ras-related C3 botulinum toxin substrate 1-small interfering RNA (Rac1-siRNA) on retinal neovascularization in a rat model.

Methods: Rac1-short hairpin RNA (shRNA) was synthesized, constructed, and transfected into HeLa cells. Reverse transcription polymerase chain reaction was then conducted to test for Rac1 gene expression. Retinal vein obstruction was performed in 25 Sprague-Dawley rats using the retinal photodynamic method. The vitrea bulbus of the eye in the shRNA rats group was transfected with the Rac1-shRNA vector, the other eye in the blank control group was transfected with the blank vector, and the interference control group was prepared by transfecting the Rac1-shRNA vector. Two weeks after transfection, the neonatal vessels were tested using fluorescein isothiocyanate-dextran retinal angiography. The number of endothelial cells beyond the internal limiting membrane was counted with hematoxylin and eosin staining.

Results: A large area of neovascularization and fluorescein isothiocyanate leakage was found in the positive control group. However, a small area of neovascularization and a little fluorescence leakage were found in the shRNA group, whereas the retinal vessels were normal in the negative control interference group. In the shRNA interference group, the mean number of endothelial cells beyond the internal limiting membrane was significantly higher than that in the positive control group or the interference negative control group (p<0.05).

Conclusions: Silencing Rac1 expression with RNA interference inhibits retinal neovascularization in rats.

Show MeSH
Related in: MedlinePlus