The effect of adenovirus-mediated recombinant Tum5 gene expression on Rhesus retinal vascular endothelial cells under high glucose_Chinese Journal of Ocular Fundus Diseases

Authors：

YangWei , ZhangYan , SunJing , 韩倩 , 贾育蓉 ,  张红

Tianjin Medical University Hospital, Tianjin Medical University Eye Institute, College of Optometry and Ophthalmology, Tianjin Medical University, Tianjin 300384, China;

Corresponding author：

张红, Email: tmuechong@sina.com

Keywords：

Endothelial cells/pathology; Cell proliferation/drug effects; Cell migration inhibition/drug effects; Gene transfer techniques; Transfection; Animal experimentation

DOI：

10.3760/cma.j.issn.1005-1015.2015.05.014

Video：

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Objective To observe the expression in vitro and the influence of adenovirus-mediated recombinant Tum5 gene to the proliferation, migration and tubing of Rhesus RF/6A cell under high glucose. Methods To construct the adenovirus vector of recombinant Tum5 gene (rAd-Tum5), and then infected RF/6A cell with it. The Flow Cytometry was used to detect the infection efficiency. RF/6A cells were divided into normal group, high glucose (HG)-control group (HG group), empty expression vector group (HG+rAd-GFP), and HG+rAd-Tum5 group. Western blot was used to detect the expression of Tum5. The CCK-8 test was applied to detect the proliferation of RF/6A cell, the Transwell test was applied to detect the migration and the Matrigel test was applied to detect the tubing of RF/6A cell under high glucose. The proliferation, migration and tubing of RF/6A were tested respectively by CCK-8 test, Transwell test and Matrigel test. Results The adenovirus vector of recombinant Tum5 gene was successfully constructed. The infection efficiency of rAd-Tum5 in RF/6A cell was 50.31% and rAd-GFP was 55.13% by the Flow Cytometry. The results of Western blot indicated that Tum5 was successfully expressed in RF/6A cell. The result of CCK-8 test, Transwell test and Matrigel test indicated that there were statistical differences between all groups in proliferation, migration and tubing of the RF/6A cell (F=44.484, 772.666, 137.696;P < 0.05). The comparison of each group indicated that the HG group was higher than normal group (P < 0.05). There were no statistical differences between HG group and HG+rAd-GFP group (P > 0.05). However, the HG+rAd-Tum5 group was less than HG group (P < 0.05), and the same to HG+rAd-GFP (P < 0.05). Conclusion The adenovirus vector of recombinant Tum5 gene can inhibit the proliferation, migration and tubing of RF/6A cell under high glucose.

Citation： YangWei, ZhangYan, SunJing. The effect of adenovirus-mediated recombinant Tum5 gene expression on Rhesus retinal vascular endothelial cells under high glucose. Chinese Journal of Ocular Fundus Diseases, 2015, 31(5): 467-471. doi: 10.3760/cma.j.issn.1005-1015.2015.05.014 Copy

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1. Maeshima Y, Manfredi M, Reimer C, et al. Identification of the anti-angiogenic site within vascular basement membrane-derived tumstatin[J]. J Biol Chem, 2001, 276(18):15240-15248.
2. Maeshima Y. Extracellular matrix-derived peptide binds to alpha vbeta 3 integrin and inhibits angiogenesis[J]. J Biol Chem, 2001, 276(34):31959-31968.
3. Maeshima Y, Sudhakar A, Lively JC, et al. Tumstatin, an endothelial cell-specific inhibitor of protein synthesis[J]. Science, 2002, 295(5552):140-143.
4. Maeshima Y. Distinct antitumor properties of a typeⅣcollagen domain derived from basement membrane[J]. J Biol Chem, 2000, 275(28):21340-21348.
5. Esipov R, Beyrakhova K, Likhvantseva V, et al. Antiangiogenic and antivascular effects of a recombinant tumstatin-derived peptide in a corneal neovascularization model[J]. Biochimie, 2012, 94(6):1368-1375.
6. Ameri H, Liu H, Liu R, et al. TWEAK/Fn14 pathway is a novel mediator of retinal neovascularization[J]. Invest Ophthalmol Vis Sci, 2014, 55(2):801-813.
7. Li Z, He T, Du K, et al. Inhibition of oxygen-induced ischemic retinal neovascularization with adenoviral 15-lipoxygenase-1 gene transfer via up-regulation of PPAR-γand down-regulation of VEGFR-2 expression. PLoS One, 2014, 9(1):85824. http://dx.plos.org/10.1371/journal.pone.0085824.
8. Mcclements ME, Maclaren RE. Gene therapy for retinal disease[J]. Translational Research, 2013, 161(4):241-254.
9. Ginn SL, Alexander IE, Edelstein ML, et al. Gene therapy clinical trials worldwide to 2012-an update[J]. J Gene Med, 2013, 15(2):65-77.
10. Maclaren RE, Groppe M, Barnard AR, et al. Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial[J]. Lancet, 2014, 383(9923):1129-1137.
11. Tannous BA, Kim DE, Fernandez JL, et al. Codon-optimized gaussia luciferase cDNA for mammalian gene expression in culture and in vivo[J]. Mol Ther, 2005, 11(3):435-443.
12. Wurdinger T, Badr C, Pike L, et al. A secreted luciferase for ex vivo monitoring of in vivo processes[J]. Nat Methods, 2008, 5(2):171-173.
13. You YJ, Xue XC, Li M, et al. Inhibition effect of pcDNA-tum-5 on the growth of S180 tumor[J]. Cytotechnology, 2008, 56(2):97-104.
14. Zhang X, Xu WR, Qian H, et al. Mesenchymal stem cells modified to express lentivirus TNF-alpha Tumstatin(45-132) inhibit the growth of prostate cancer[J]. J Cell Mol Med, 2011, 15(2):433-444.

Chinese Journal of Ocular Fundus Diseases

The effect of adenovirus-mediated recombinant Tum5 gene expression on Rhesus retinal vascular endothelial cells under high glucose

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