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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 / 中华眼底病杂志
Article in Chinese | WPRIM | ID: wpr-711972
Responsible library: WPRO
ABSTRACT
Objective To 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,a-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.Results The 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).Conclusion The success in producing CTGF shRNA lentivirus particle suggests that CTGF shRNA lentivirus can effectively knock down CTGF expression.

Full text: Available Index: WPRIM (Western Pacific) Language: Chinese Journal: Chinese Journal of Ocular Fundus Diseases Year: 2018 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Language: Chinese Journal: Chinese Journal of Ocular Fundus Diseases Year: 2018 Type: Article