关键词: 血管内皮细胞, 人脐静脉内皮细胞, SV40, 大T抗原, CD31, Cas9

Abstract: Objective To establish primary and simian virus 40 (SV40) T antigen transformed human vascular endothelial cell models, and provide available resources for endothelial research. Methods Human umbilical vein endothelial cells(HUVEC), human umbilical artery endothelial cells(HUAEC), great saphenous vein endothelial cells(GSVEC) and endothelial cells form endometrium and liver tissue were isolated and cultured respectively. Then, the primary endothelial cells were transformed by lentivirus containing SV40 big T and small T antigens, and continuously subcultured in vitro. The expression of CD31 was detected by flow cytometry, species identificationand mycoplasma detection by PCR, and cell identity was identified by STR detection. The transformed ECs were checked for HLA types. Some of them were tested for RNA expression profile and infected by Cas9 lentivirus to establish stable clones. Results Totally 187 cell lines of transformed HUVEC, 1 of transformed HUAEC, 5 of transformed GSVEC, 1 of transformed endothelial cells from endometrium and 1 of transformed endothelial cells from liver tissue, and 9 monoclonal HUVEC cell lines stably expressing Cas9 protein were established. All the transformed umbilical endothelial cells were CD31 positive ranging from 20%-90% for 20 cases, while for the rest 168 cases the positive rate was more than 90%. RNA expression revealed stable activation of cell proliferation(cell cycle and DNA synthesis). Their species were identified as human origin. The STR results were consistent with those of the primary culture and unique, and there was no mycoplasma contamination. All these cells could be obtained with the sharing services of National Science and Technology Infrastructure, the National Biomedical Cell-line Resource centers(NSTI-BMCR). Conclusions A series of primary and SV40 T antigen transformed human vascular endothelial cell models have been established, which provide a tool for the study of cardiovascular diseases, inflammation, tumors and immune-related diseases.

Key words: vascular endothelial cells, human umbilical vein endothelial cells, SV40, LT antigen, CD31, Cas9