ABSTRACT
Objective: To construct the RHBDF2 gene over-expression lentivirus vector and to establish the KA. hy926 cells stably expressing RHBDF2, and to provide the evidence for the construction of RHBDF2 gene over-expression lentivirus vector and the establishment of RHBDF2 cells stably expressing RHBDF2. Methods: According to the sequence of RHBDF2 gene provided by NCBI, and the primers were designed and synthesized; the RHBDF2 gene was amplified by PCR method, and the target gene was cloned into the entry vector by Gateway cloning technology, and then subcloned into the lentivirus vector pLV [Exp]-EGFP to construct the recombinant lentivirus plasmid pLV I Exp]-EGFP-RHBDF2; the lentivirus expression vector plasmid pLV I Exp]-EGFP and the recombinant lentivirus plasmid pLV I Exp]-EGFP-RHBDF2 were co-transfected into the HEK293T cells with the virus-assisted packaging plasmids to package the lentivirus and the titer of the lentivirus was detected. The EA. hy926 cells infected with pLV [Exp]-EGFP-control were used as control group and the EA. hy926 cells infected with pLV [Exp]-EGFP-RHBDF2 were used as experiment group. The EA. hy926 cells stably expressing RHBDF2 were screened by puromycin. The fluorescent quantitative PCR (qPCR) and Western blotting methods were used to detect the expression levels of RHBDF2 mRNA and protein in the EA. hy926 cells in control group and experiment group. Results: The enzyme digestion electrophoresis and sequencing results showed that the gene sequence of the EA. hy926 cells over-expression lentivirus vector in experiment group was completely consistent with the designed and synthesized sequence. The lentivirus titer in control group was 1 X 10 TU • mL , and the lentivirus titer in experiment group was 3X 10 TU • mL . The EA. hy926 cells were successfully infected with the lentivirus under fluorescence microscope and the infection efficiency was above 95%. The qPCR detection results showed that the expression level of RHBDF2 mRNA in the EA. hy926 cells in experiment group was higher than that in control group (P'<0.01). The Western blotting results showed that the expression level of RHBDF2 protein in the EA. hy926 cells in experiment group was higher than that in control group ( P < 0. 05 ). Conclusion: The lentivirus vector over-expressing RHBDF2 is successfully constructed∗ and the EA. hy926 cell line stably up-regulating the expression of RHBDF2 is established by using pLV [Exp]-EGFP-RHBDF2 lentivirus.
ABSTRACT
Objective: To investigate the effect and mechanism of cinnamaldehyde on the angiogenesis of diabetic retinopathy, and the effect of cinnamaldehyde on vascular endothelial growth factor (VEGF) induced proliferation, migration, tube formation and Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway of EA.hy 926 cells were observed. Method:EA.hy 926 cells were divided into normal control group, model group (7 μg·L-1 VEGF), and VEGF+cinnamaldehyde group (60, 90, 120, 150 μmol·L-1). The methyl thiazolyl tetrazolium (MTT) assay and scratch test were used to observe the effect of cinnamaldehyde on the proliferation and migration of EA. hy 926 cells induced by VEGF. EA. hy 926 cells were divided into normal control group, model group (7 μg·L-1 VEGF), and VEGF+cinnamaldehyde group (90, 150 μmol·L-1). The tube formation experiment was used to observe the effect of cinnamaldehyde on the tube formation of EA. hy 926 cells induced by VEGF. EA. hy 926 cells were divided into normal control group, model group (7 μg·L-1 VEGF), VEGF+AG490 group (50 μmol·L-1), VEGF+cinnamaldehyde group (90 μmol·L-1), VEGF+cinnamaldehyde group (150 μmol·L-1), and VEGF+cinnamaldehyde group (150 μmol·L-1)+AG490 group (50 μmol·L-1). Western Blot method was used to explore the effect of cinnamaldehyde on the JAK2/STAT3 signaling pathway in EA.hy 926 cells induced by VEGF. Result:Compared with the control group, model group obviously promoted the proliferation and migration of EA.hy 926 cells(P-1) significantly suppressed VEGF-induced proliferation and migration of EA.hy 926 cells (P-1) showed an obvious inhibitory effect on the number of nodes, junctions and meshes of tubules (PPPP-1) significantly reduced the expressions of P-JAK2, P-STAT3, STAT3 proteins (P-1) obviously reduced the expressions of p-STAT3 and STAT3 proteins (PPConclusion:Cinnamaldehyde showed a significantly inhibitory effect on the proliferation, migration and tube formation of VEGF-induced EA.hy 926 cells, which was related to the inhibition of the activation of JAK2/STAT3 pathway.