Construction of connective tissue growth factor recombinant interference vector lentiviral particle and its inhibitory effect on endogenous connective tissue growth factor expression in retinal vascular endothelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

Niu Rui , Dong Lijie , Ma Teng , Du Xueli , He Yanhua , Cui Weina ,  Hu Bojie

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

Corresponding author：

Hu Bojie, Email: bhu07@tmu.edu.cn

Keywords：

Connective tissue growth factor; Lentivirus infections; Retinal Vessels/cytology; Endothelial cells

DOI：

10.3760/cma.j.issn.1005-1015.2018.06.011

Video：

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ObjectiveTo construct the connective tissue growth factor (CTGF) recombinant interference vector (shRNA) and observe its inhibitory effect on the expression of endogenous CTGF in retinal vascular endothelial cells. Methods The human CTGF shRNA was constructed and the high-titer CTGF shRNA lentivirus particles was acquired via three-plasmid lentivirus packaging system to infect retinal vascular endothelial cells. The optimal multiplicity and onset time of lentivirus infection were identified by tracing down the red florescent protein in interference vector. The cells were classified into three groups: blank control group, infection control group and CTGF knockdown group. The differences in cells migrating ability was observed through Transwell allay. The mRNA and protein expression of CTGF, fibronectin, α-smooth muscle actin (α-SMA) and collagen Ⅰ (Col Ⅰ) were quantified through real-time PCR testing and Western blot system. Data between the three groups were examined via one-way analysis of variance. ResultsThe result showed that an optimal multiplicity of 20 and onset time of 72 hours were the requirements to optimize lentivirus infection. Transwell allay result showed a contrast in the number of migrated cells in the CTGF knockdown group and that in the blank control group and infection control group (F=20.64, P=0.002). Real-time PCR testing showed a contrast in related gene expression (CTGF, fibronectin, α-SMA and Col Ⅰ) in the CTGF knocked-down group and that in the blank control group and infection control group (F=128.83, 124.44, 144.76, 1 374.44; P=0.000, 0.000, 0.000, 0.000). Western blot system showed the statistical significance of the contrasted number of related protein expression (CTGF, fibronectin, α-SMA and Col Ⅰ) in the knockdown group and that in the blank control group (F=22.55, 41.60, 25.73, 161.68; P=0.002, 0.000, 0.001, 0.000). ConclusionThe success in producing CTGF shRNA lentivirus particle suggests that CTGF shRNA lentivirus can effectively knock down CTGF expression.

Citation： Niu Rui, Dong Lijie, Ma Teng, Du Xueli, He Yanhua, Cui Weina, Hu Bojie. Construction of connective tissue growth factor recombinant interference vector lentiviral particle and its inhibitory effect on endogenous connective tissue growth factor expression in retinal vascular endothelial cells. Chinese Journal of Ocular Fundus Diseases, 2018, 34(6): 580-585. doi: 10.3760/cma.j.issn.1005-1015.2018.06.011 Copy

1.	Asrar AAE, Struyf S, Opdenakker G, et al. Expression of stem cell factor/c-kit signaling pathway components in diabetic fibrovascular epiretinal membranes[J]. Mol Vis, 2010, 16: 1098-1107.
2.	Parikh RN, Traband A, Kolomeyer AM, et al. Intravitreal bevacizumab for the treatment of vitreous hemorrhage due to proliferative diabetic retinopathy[J]. Am J Ophthalmol, 2017, 176: 194-202. DOI: 10.1016/j.ajo.2017.01.010.
3.	Wei Q, Zhang T, Jiang R, et al. Vitreous fibronectin and fibrinogen expression increased in eyes with proliferative diabetic retinopathy after intravitreal anti-VEGF therapy[J]. Invest Ophthalmol Vis Sci, 2017, 58(13): 5783-5791. DOI: 10.1167/iovs.17-22345.
4.	Klaassen I, van Geest RJ, Kuiper EJ. The role of CTGF in diabetic retinopathy[J]. Exp Eye Res, 2015, 133: 37-48. DOI: 10.1016/j.exer.2014.10.016.
5.	胡博杰, 曾勍, 刘新玲, 等. Avastin玻璃体腔注射后糖尿病视网膜病变增生膜中细胞因子的变化[J]. 中华实验眼科杂志, 2013, 31(1): 55-59. DOI: 10.3760/cma.j.issn.2095-0160.2013.01.013.Hu BJ, Zeng Q, Liu XL et al. Influence of intravitreal avastin on the expression of cell factors in retinal proliferative membrane in proliferative diabetic retinopathy eye[J].Chin J Exp Ophthalmol, 2013, 31(1): 55-59. DOI: 10.3760/cma.j.issn.2095-0160.2013.01.013.
6.	Hu B, Yan Z, Zeng Q, et al. Intravitreal injection of ranibizumab and CTGFshRNA improves retinal gene expression and microvessel ultrastructure in a rodent model of diabetes[J]. Int J Mol Sci, 2014, 15(1): 1606-1624. DOI: 10.3390/ijms15011606.
7.	Kuiper EJ, Van Nieuwenhoven FA, de Smet M D, et al. The angio-fibrotic switch of VEGF and CTGF in proliferative diabetic retinopathy[J/OL]. PLoS One, 2008, 3(7): 2675[2008-07-16]. http://dx.plos.org/10.1371/journal.pone.0002675. DOI: 10.1371/journal.pone.0002675.
8.	Van Geest RJ, Klaassen I, Lesnik-Oberstein SY, et al. Vitreous TIMP-1 levels associate with neovascularization and TGF-β2 levels but not with fibrosis in the clinical course of proliferative diabetic retinopathy[J]. J Cell Commun Signal, 2013, 7(1): 1-9. DOI: 10.1007/s12079-012-0178-y.
9.	Van Geest RJ, Lesnik-Oberstein SY, Tan HS, et al. A shift in the balance of vascular endothelial growth factor and connective tissue growth factor by bevacizumab causes the angiofibrotic switch in proliferative diabetic retinopathy[J]. Br J Ophthalmol, 2012, 96(4): 587-590. DOI: 10.1136/bjophthalmol-2011-301005.
10.	Hendriks WT, Ruitenberg MJ, Blits B, et al. Viral vector-mediated gene transfer of neurotrophins to promote regeneration of the injured spinal cord[J]. Prog Brain Res, 2004, 146: 451-476. DOI: 10.1016/S0079-6123(03)46029-9.
11.	Bo X, Wu D, Yeh J, et al. Gene therapy approaches for neuroprotection and axonal regeneration after spinal cord and spinal root injury[J]. Curr Gene Ther, 2011, 11(2): 101-115. DOI: 10.2174/156652311794940773.
12.	Chira S, Jackson CS, Oprea I, et al. Progresses towards safe and efficient gene therapy vectors[J]. Oncotarget, 2015, 6(31): 30675-30703. DOI: 10.18632/oncotarget.5169.
13.	杨幼萍, 丁燕, 王继荣, 等. 整合素连接激酶基因敲降和黑色素瘤分化相关基因过表达慢病毒载体构建和鉴定[J]. 浙江大学学报(医学版), 2014, 43(2): 193-199. DOI: 10.3785/j.issn.1008-9292.2014.03.013.Yang YP, Ding Y, Wang JR, et al. Construction and identification of lentiviral vector containing human ILK-shRNA and mda7 gene[J].Journal of Zhengjiang University (Medical Science), 2014, 43(2): 193-199. DOI: 10.3785/j.issn.1008-9292.2014.03.013.
14.	Winkler JL, Kedees MH, Guz Y, et al. Inhibition of connective tissue growth factor by small interfering ribonucleic acid prevents increase in extracellular matrix molecules in a rodent model of diabetic retinopathy[J]. Mol Vis, 2012, 18: 874-886.
15.	Coral K, Angayarkanni N, Madhavan J, et al. Lysyl oxidase activity in the ocular tissues and the role of LOX in proliferative diabetic retinopathy and rhegmatogenous retinal detachment[J]. Invest Ophthalmol Vis Sci, 2008, 49(11): 4746-4752. DOI: 10.1167/iovs.07-1550.
16.	Morishige N, Uemura A, Morita Y, et al. Promotion of corneal epithelial wound healing in diabetic rats by the fibronectin-derived peptide PHSRN[J]. Cornea, 2017, 36(12): 1544-1548. DOI: 10.1097/ICO.0000000000001344.
17.	Ina K, Kitamura H, Tatsukawa S, et al. Significance of α-SMA in myofibroblasts emerging in renal tubulointerstitial fibrosis[J]. Histol Histopathol, 2011, 26(7): 855-866. DOI: 10.14670/HH-26.855.
18.	Li X, Wu Z, Ni J, et al. Cathepsin B regulates collagen expression by fibroblasts via prolonging TLR2/NF-κB activation[J/OL]. Oxid Med Cell Longev, 2016, 2016: 7894247[2016-08-28]. https://dx.doi.org/10.1155/2016/7894247. DOI: 10.1155/2016/7894247.
19.	戎慧丰, 颜华.增生型糖尿病视网膜病变纤维化相关因子的研究进展[J].中华实验眼科杂志, 2011, 29(5): 473-476. DOI: 10.3760/cma.j.issn.2095-0160.2011.05.020.Rong HF, Yan H. Research progress in correlation factor on fibrosis of proliferative diabetic retinopathy[J].Chin J Exp Ophthalmol, 2011, 29(5): 473-476. DOI: 10.3760/cma.j.issn.2095-0160.2011.05.020.
20.	Altrock E, Sens C, Wuerfel C, et al. Inhibition of fibronectin deposition improves experimental liver fibrosis[J]. J Hepatol, 2015, 62(3): 625-633. DOI: 10.1016/j.jhep.2014.06.010.
21.	Wang Y, Lin C, Ren Q, et al. Astragaloside effect on TGF-β1, SMAD2/3, and α-SMA expression in the kidney tissues of diabetic KKAy mice[J]. Int J Clin Exp Pathol, 2017, 8(6): 6828-6834.
22.	牛瑞, 东莉洁, 马腾, 等.抗血管内皮生长因子药物治疗后视网膜血管内皮细胞基因表达谱的RNA-Seq分析[J].中华眼底病杂志, 2018, 34(3): 275-280. DOI: 10.3760/cma.j.issn.1005-1015.2018.03.016.Niu R, Dong LJ, Ma T, et al. RNA-Seq analysis of gene expression profiling in human retinal vascular endothelial cells after anti-vascular endothecial growth factor treatment[J].Chin J Ocul Fundus Dis, 2018, 34(3): 275-280. DOI: 10.3760/cma.j.issn.1005-1015.2018.03.016.

1. Asrar AAE, Struyf S, Opdenakker G, et al. Expression of stem cell factor/c-kit signaling pathway components in diabetic fibrovascular epiretinal membranes[J]. Mol Vis, 2010, 16: 1098-1107.
2. Parikh RN, Traband A, Kolomeyer AM, et al. Intravitreal bevacizumab for the treatment of vitreous hemorrhage due to proliferative diabetic retinopathy[J]. Am J Ophthalmol, 2017, 176: 194-202. DOI: 10.1016/j.ajo.2017.01.010.
3. Wei Q, Zhang T, Jiang R, et al. Vitreous fibronectin and fibrinogen expression increased in eyes with proliferative diabetic retinopathy after intravitreal anti-VEGF therapy[J]. Invest Ophthalmol Vis Sci, 2017, 58(13): 5783-5791. DOI: 10.1167/iovs.17-22345.
4. Klaassen I, van Geest RJ, Kuiper EJ. The role of CTGF in diabetic retinopathy[J]. Exp Eye Res, 2015, 133: 37-48. DOI: 10.1016/j.exer.2014.10.016.
5. 胡博杰, 曾勍, 刘新玲, 等. Avastin玻璃体腔注射后糖尿病视网膜病变增生膜中细胞因子的变化[J]. 中华实验眼科杂志, 2013, 31(1): 55-59. DOI: 10.3760/cma.j.issn.2095-0160.2013.01.013.Hu BJ, Zeng Q, Liu XL et al. Influence of intravitreal avastin on the expression of cell factors in retinal proliferative membrane in proliferative diabetic retinopathy eye[J].Chin J Exp Ophthalmol, 2013, 31(1): 55-59. DOI: 10.3760/cma.j.issn.2095-0160.2013.01.013.
6. Hu B, Yan Z, Zeng Q, et al. Intravitreal injection of ranibizumab and CTGFshRNA improves retinal gene expression and microvessel ultrastructure in a rodent model of diabetes[J]. Int J Mol Sci, 2014, 15(1): 1606-1624. DOI: 10.3390/ijms15011606.
7. Kuiper EJ, Van Nieuwenhoven FA, de Smet M D, et al. The angio-fibrotic switch of VEGF and CTGF in proliferative diabetic retinopathy[J/OL]. PLoS One, 2008, 3(7): 2675[2008-07-16]. http://dx.plos.org/10.1371/journal.pone.0002675. DOI: 10.1371/journal.pone.0002675.
8. Van Geest RJ, Klaassen I, Lesnik-Oberstein SY, et al. Vitreous TIMP-1 levels associate with neovascularization and TGF-β2 levels but not with fibrosis in the clinical course of proliferative diabetic retinopathy[J]. J Cell Commun Signal, 2013, 7(1): 1-9. DOI: 10.1007/s12079-012-0178-y.
9. Van Geest RJ, Lesnik-Oberstein SY, Tan HS, et al. A shift in the balance of vascular endothelial growth factor and connective tissue growth factor by bevacizumab causes the angiofibrotic switch in proliferative diabetic retinopathy[J]. Br J Ophthalmol, 2012, 96(4): 587-590. DOI: 10.1136/bjophthalmol-2011-301005.
10. Hendriks WT, Ruitenberg MJ, Blits B, et al. Viral vector-mediated gene transfer of neurotrophins to promote regeneration of the injured spinal cord[J]. Prog Brain Res, 2004, 146: 451-476. DOI: 10.1016/S0079-6123(03)46029-9.
11. Bo X, Wu D, Yeh J, et al. Gene therapy approaches for neuroprotection and axonal regeneration after spinal cord and spinal root injury[J]. Curr Gene Ther, 2011, 11(2): 101-115. DOI: 10.2174/156652311794940773.
12. Chira S, Jackson CS, Oprea I, et al. Progresses towards safe and efficient gene therapy vectors[J]. Oncotarget, 2015, 6(31): 30675-30703. DOI: 10.18632/oncotarget.5169.
13. 杨幼萍, 丁燕, 王继荣, 等. 整合素连接激酶基因敲降和黑色素瘤分化相关基因过表达慢病毒载体构建和鉴定[J]. 浙江大学学报(医学版), 2014, 43(2): 193-199. DOI: 10.3785/j.issn.1008-9292.2014.03.013.Yang YP, Ding Y, Wang JR, et al. Construction and identification of lentiviral vector containing human ILK-shRNA and mda7 gene[J].Journal of Zhengjiang University (Medical Science), 2014, 43(2): 193-199. DOI: 10.3785/j.issn.1008-9292.2014.03.013.
14. Winkler JL, Kedees MH, Guz Y, et al. Inhibition of connective tissue growth factor by small interfering ribonucleic acid prevents increase in extracellular matrix molecules in a rodent model of diabetic retinopathy[J]. Mol Vis, 2012, 18: 874-886.
15. Coral K, Angayarkanni N, Madhavan J, et al. Lysyl oxidase activity in the ocular tissues and the role of LOX in proliferative diabetic retinopathy and rhegmatogenous retinal detachment[J]. Invest Ophthalmol Vis Sci, 2008, 49(11): 4746-4752. DOI: 10.1167/iovs.07-1550.
16. Morishige N, Uemura A, Morita Y, et al. Promotion of corneal epithelial wound healing in diabetic rats by the fibronectin-derived peptide PHSRN[J]. Cornea, 2017, 36(12): 1544-1548. DOI: 10.1097/ICO.0000000000001344.
17. Ina K, Kitamura H, Tatsukawa S, et al. Significance of α-SMA in myofibroblasts emerging in renal tubulointerstitial fibrosis[J]. Histol Histopathol, 2011, 26(7): 855-866. DOI: 10.14670/HH-26.855.
18. Li X, Wu Z, Ni J, et al. Cathepsin B regulates collagen expression by fibroblasts via prolonging TLR2/NF-κB activation[J/OL]. Oxid Med Cell Longev, 2016, 2016: 7894247[2016-08-28]. https://dx.doi.org/10.1155/2016/7894247. DOI: 10.1155/2016/7894247.
19. 戎慧丰, 颜华.增生型糖尿病视网膜病变纤维化相关因子的研究进展[J].中华实验眼科杂志, 2011, 29(5): 473-476. DOI: 10.3760/cma.j.issn.2095-0160.2011.05.020.Rong HF, Yan H. Research progress in correlation factor on fibrosis of proliferative diabetic retinopathy[J].Chin J Exp Ophthalmol, 2011, 29(5): 473-476. DOI: 10.3760/cma.j.issn.2095-0160.2011.05.020.
20. Altrock E, Sens C, Wuerfel C, et al. Inhibition of fibronectin deposition improves experimental liver fibrosis[J]. J Hepatol, 2015, 62(3): 625-633. DOI: 10.1016/j.jhep.2014.06.010.
21. Wang Y, Lin C, Ren Q, et al. Astragaloside effect on TGF-β1, SMAD2/3, and α-SMA expression in the kidney tissues of diabetic KKAy mice[J]. Int J Clin Exp Pathol, 2017, 8(6): 6828-6834.
22. 牛瑞, 东莉洁, 马腾, 等.抗血管内皮生长因子药物治疗后视网膜血管内皮细胞基因表达谱的RNA-Seq分析[J].中华眼底病杂志, 2018, 34(3): 275-280. DOI: 10.3760/cma.j.issn.1005-1015.2018.03.016.Niu R, Dong LJ, Ma T, et al. RNA-Seq analysis of gene expression profiling in human retinal vascular endothelial cells after anti-vascular endothecial growth factor treatment[J].Chin J Ocul Fundus Dis, 2018, 34(3): 275-280. DOI: 10.3760/cma.j.issn.1005-1015.2018.03.016.

Chinese Journal of Ocular Fundus Diseases

Construction of connective tissue growth factor recombinant interference vector lentiviral particle and its inhibitory effect on endogenous connective tissue growth factor expression in retinal vascular endothelial cells

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