NFKB1 Gene Polymorphism Implicates NF-κB Signal Pathway And Apoptosis During Oxidative Damage In HUVECs

Objective: To explore effect of NFKB1 gene polymorphism(-94ins/del ATTG) to NF-kB signal pathway and apoptosis during oxidative damage in primary human umbilical vein endothelial cells(HUVECs). Methods: 1) Isolate and culture HUVECs, identify their purity with vWF and CD31 antibody by immunofluorescence and flow cytometry. 2) Identify the polymorphism of NFKB1 gene to three genotype(DD, ID and II genotype) by polymerase chain reaction- restriction fragment length polymorphism(PCR-RFLP). 3) Explore p50 and p65 level by Western blot and immunofluorescence. 4) Explore cells viability by CCK8 to establish the oxidative injury model. 5) Explore p50 and p65 level in oxidative injury model by Western blot and immunofluorescence in oxidative injury model. 6) Explore apoptosis level in oxidative injury model by Western blot and flow cytometry in oxidative injury model. Result: 1) 63 HUVECs were Isolated and cultured, immunofluorescence analysis showed that the expression of vWF antibody was 98±0.12%, flow cytometry analysis showed that the expression of CD 31 antibody was 93.3%; 2) The result of PCR-RFLP showed that II was 13, DD was 13, ID was 37; 3) Western blotting and immunofluorescence showed that the protein levels of P50 in II were notably increased than which in DD; 4) The result of CCK-8 showed that HUVECs induced by H2O2 was 200 umol/L and 12 h; 5) Western blotting and immunofluorescence showed that H2O2 could activate the NF-κB signal pathway, and the nuclear protein levels of P50 and P 65 in II were notably increased than which in DD; 6) Western blotting and flow cytometry analysis showed that H2O2 could enhance HUVECs apoptosis, and the levels of apoptosis in DD were notably increased than which in II. Conclusion: 1) Isolated and cultured HUVECs can obtain high pure HUVECs, and effective, safe and precise; 2) NFKB1 gene polymorphism could effect the protein levels of P50 in NF-κB signal pathway; 3) H2O2 could inhibit the viability of HUVECs, and establish oxidative stress model; 4) H2O2 could induce HUVECs and activate NF-κB signal pathway, and NFKB1 gene polymorphism could effect the level of NF-κB signal pathway; 5) H2O2 could enhance HUVECs apoptosis, and NFKB1 gene polymorphism could effect the level of apoptosis.