RESUMEN
Objective To construct a human renal epithelial cell line HEK293T by CRISPR-Cas9-based site-directed knock-in of vascular endothelial growth factor 165 (VEGF165) gene, and avoid the off-target effect caused by lentivirus infection. Methods The VEGF165 expression vector with homologous arm (pUCm-T-VEGF165 plasmid) and the sgRNA expression vector [pSpCas9(BB)-2A-Puro-sgRNA plasmid] were designed and constructed based on the DNA sequence of the EZH2 gene, and then co-transfected into HEK293T cells. The expression of VEGF165 mRNA was detected by qPCR and the expressions of VEGF165 proteins were detected by Western Blot. Results The qPCR and Western Blot results showed that, comparing with the control, the pUCm-T-VEGF165 plasmid and pSpCas9(BB)-2A-Puro-sgRNA plasmid, the expression of the co-transfection plasmid were significantly increased, i.e. 3.42±0.30 vs. 1.02±0.21, 1.13±0.16 and 0.98±0.18 for the VEGF165 mRNA level (all P<0.01), and 1.13±0.16 vs. 1.02±0.06, 0.88±0.03 and 0.80±0.05 for the VEGF165 protein level (all P<0.01), respectively. Besides, the expression of EZH2 was significantly down-regulated, i.e. 0.14±0.06 vs. 1.08±0.11, 1.02±0.12 and 1.13±0.16 for the EZH2 mRNA level (all P<0.01), and 0.23±0.03 vs. 1.05±0.13, 0.91±0.04 and 0.81±0.06 for the EZH2 protein level (all P<0.01), respectively. This result showed that the VEGF165 was successfully inserted into the EZH2 genome, interfering the EZH2 expression. Conclusions VEGF165 gene can be successfully knocked into HEK293T cells by CRISPR/Cas9 system.
RESUMEN
Objective To study on the role of histone methylation enzyme enhancer of zeste homolog 2 (EHZ2) and vascular endothelial growth factor 165 (VEGF165) in momymoya disease. Methods The animal model of moyamoya disease was established by ear vein injection of horse serum in New Zealand rabbits. VEGF165 was over-expressed in situ by packaging lentivirus. Real-time quantitative PCR and Western Blot were used to detect the expression of VEGF165, EZH2 and H3K27me3 in the brain tissues of the animal models. Results Compared with the normal control group, the expression levels of mRNA and protein of EZH2 in the moyamoya disease model group were increased (EZH2 mRNA:P<0.01), and the level of histone H3K27me3 was increased. After overexpression of VEGF165 in the moyamoya disease model group, the expression levels of mRNA and protein of EZH2 was further increased (EZH2 mRNA: P<0.01), and the level of histone H3K27me3 was also increased. Conclusions EZH2 plays a certain role in the pathogenesis of moyamoya disease, and the expression of EZH2 is regulated by VEGF 165, which provides a theoretical basis for the study of the pathogenesis of moyamoya disease.
RESUMEN
Objective:To study the effect of vascular endothelial growth factor 165b (VEGF165b) on the biological characteristics of human hepatocellular carcinoma HepG2 cells,and to explore its mechanisr.Methods;The HepG2 cells were divided into blank group (treated with transfection reagent),control group (transfected with pcDNA3.0 expression vector) and pcDNA-VEGF165b group (transfected with pcNDA-VEGF165b).MTT assay was used to detect the survival rate of HepG2 cells;RT-PCR and Western blotting method were used to detect the expression levels of VEGF165b and VEGF165 mRNA and protein in the HepG2cells;Transwell chamber assay was used to measure the migration ability of HepG2 cells.Results:Compared with blank group,there was no significant changes in the expression levels of VEGF165b and VEGF165 mRNA and protein in the HepG2 cells in control group (P＞0.05).Compared with blank group,the expression levels of VEGF165b mRNA and protein in the HepG2 cells in PcDNA-VEGF165b group were significantly increased (P＜0.05),and the expression levels of VEGF165 mRNA and protein were significantly decreased (P＜0.05).There was no significant difference in the survival rates between blank group and control group (P＞0.05).The survival rate of HepG2 cells in PcDNA-VEGF165b group was lower than those in blank group and control group,but the differences were not statistically significant (P＞0.05).The cell migration experiment results showed that there was no significant difference in the migration rate between blank group and control group (P＜0.05).Compared with blank group and control group,the migration rate of HepG2 cells in PcDNA-VEGF165b group was significantly reduced (P＜ 0.05).Conclusion:VEGF165b can inhibit the expressions of VEGF165 mRNA and protein,and VEGF165b has no effect on the proliferation of hepatocellular carcinoma cells;but it can reduce the migration of hepatocellular carcinoma cells.
RESUMEN
Objective: To study the effect of vascular endothelial growth factor 165b (VEGF165b) on the biological characteristics of human hepatocellular carcinoma HepG2 cells, and to explore its mechanism. Methods: The HepG2 cells were divided into blank group (treated with transfection reagent), control group (transfected with pcDNA3. 0 expression vector) and pcDNA-VEGF165b group (transfected with pcNDA-VEGF165b). MTT assay was used to detect the survival rate of HepG2 cells; RT-PCR and Western blotting method were used to detect the expression levels of VEGF165b and VEGF165 mRNA and protein in the HepG2 cells; Transwell chamber assay was used to measure the migration ability of HepG2 cells. Results: Compared with blank group, there was no significant changes in the expression levels of VEGF165b and VEGF165 mRNA and protein in the HepG2 cells in control group (P>0. 05). Compared with blank group, the expression levels of VEGF165b mRNA and protein in the HepG2 cells in PcDNA-VEGF165b group were significantly increased (P0. 05). The survival rate of HepG2 cells in PcDNA-VEGF165b group was lower than those in blank group and control group, but the differences were not statistically significant (P>0. 05). The cell migration experiment results showed that there was no significant difference in the migration rate between blank group and control group (P<0. 05). Compared with blank group and control group, the migration rate of HepG2 cells in PcDNA-VEGF165b group was significantly reduced (P<0.05). Conclusion: VEGF165b can inhibit the expressions of VEGF165 mRNA and protein, and VEGF165b has no effect on the proliferation of hepatocellular carcinoma cells; but it can reduce the migration of hepatocellular carcinoma cells.
RESUMEN
[Objective]To construct the recombinant adenovirus vector co-expressing human vascular endothelial growth factor 165(VEGF165) and angiogenin-1(Ang-1),and to observe the expression of target gene after transfection.[Method]Molecular biologic techniques were used to clone the genes of VEGF165 and Ang-1,and PCR was used to amplify the DNA sequence of IRES(internal ribozyme entry site) from pIRES2-EGFP.The genes of VEGF165,IRES and Ang-1 were subcloned to pTrack-CMV one by one to get the plasmid named pTrack-CMV-VIA,which contained all the three genes.The linearized shuttle plasmid was cotransformed into BJ5183 bacteria with backbone vector AdEasy-1.Further the recombinant plasmid was packaged and amplified in QBI-293A cells after Pac I digestion to get adenovirus vector pAd-VIA.The expression of the gene of interests was evaluated by fluorescence microscopic analysis of GFP expression and enzyme linked immunosorbent assay(ELISA).[Result]The recombinant adenovirus plasmid was consistent with that shown by sequencing and restriction endonucleases digestion.The recombinant pAd-VIA vector was packaged and amplified successfully in QBI-293A cells.The titer was 2?1010 PFU/ml after amplifying.High positive GFP was expressed in the field through fluorescence microscopy after being transfected by recombinant pAd-VIA.ELISA indicated the expression of the target genes.The difference between the tansfected and non-transfected group was significant(P
RESUMEN
Objective To obtain skin seed cells which can highly express active vascular endothelial growth factor 165(VEGF165) so as to promote refractory wound healing by gene therapy in combination with cell engineering.Methods After pIRES2-EGFP-hVEGF165 was transfected into dermal mesenchymal stem cells(DMSCs) by lipofectin,the expression of VEGF mRNA was detected by RT-PCR,VEGF protein in the supernatant and in the cells were assayed by enzyme-linked immunosorbent assay(ELISA) and Westen blotting respectively.To evaluate the activity of VEGF secreted by transfected DMSCs,hECV304 was cultured with the supernatant of transfected DMSCs and its proliferation activity was analyzed by MTT assays.Meanwhile,the proliferation activity of VEGF-transfected DMSCs and non transfected DMSCs was investigated by MTT.Results The results of RT-PCR,ELISA and Western blot demonstrated that the expression of VEGF in transfected DMSCs was about 1.6 times than that of control DMSCs.The product not only enhanced the proliferation of hECV304 but also increased the proliferation of transfected DMSCs.Conclusion The plasmid pIRES2-EGFP-hVEGF165 is successfully transfected into DMSCs with the aid of lipotransfection,and hVEGF165-transfected DMSCs might high-efficiently secrete highly active VEGF165.
RESUMEN
Objective:To investigate the differential expression of VEGF165b in the renal cell carcinoma (RCC) and normal renal tissues and its role in the development of RCC. Methods: S-P immunohistochemistry was used to detect the expression of VEGF165b protein in 30 specimens of paraffin-embedded RCC tissues and 29 specimens of paraffin-embedded normal renal tissues. RT-PCR was performed to detect the expression of VEGF165b mRNA in 32 specimens of fresh RCC tissues and 30 specimens of fresh normal renal tissues. Results: Among 29 of normal renal tissues,28 specimens had positive expression of VEGF165b protein,with the positive rate of 96.55%(28/29) that was significantly higher than 20.00%(6/30)in RCC tissues(P