| Objective:To investigate the methods of isolation in vitro, culture, cryopreservation, resuscitation and identification, establish a practical modle and experimental means for Vascular Endothelial Cells; detect the relative expression levels of estrogen receptor (ERa and ERβ) in HUVECs; then study the influence of ERa and ERP on the initial process of Atherosclerosis—the adhesion between monocytes (THP-1) and HUVECs.Methods:Cut the newborn umbilical cord under sterile conditions (20cm～30cm), successfully isolate, culture Human Umbilical Vein Endothelial Cells in vitro by collagenaseⅡdigestion method. It was not digested with trypsin until the cultured cells reached confluence over 80%. Regularly frozen and recovery the cultured cells to maintain the same generation of homologous. Respectively, identify the cultured cells by cell morphology,Ⅷfactor related antigen immunohistochemistry and the detection of surface antigen CD34. Follow-up experiments with 2～4 generations of HUVECs, detect the expression levels of estrogen receptor (ERαand ERβ) in HUVECs by the two methods --- western blot and RT-PCR from protein and mRNA levels. The plasmids (pWPI-flag-hERα, pWPI-flag-ERβ, PsuperiorERαsiRNA, PsuperiorERsiRNA, pWPI-vector, pLVTHM-scramble) were transfected into HUVECs by Lipofectamine 2000, after 48 hours, co-cultured 1.5h with the same number of THP-1 (green fluorescent staining), divided into six groups:ERαoverexpress Group:HUVEC(ERα+) and THP-1, ERαknockdown group:HUVEC (ERα-) and THP-1; ERβoverexpress Group:HUVEC (ERβ+) and THP-1; ERβknockdown group:HUVEC (ERβ-) and THP-1; Vector group:HUVEC (Ve) and THP-1;Scramble group:HUVEC (Sc) and THP-1, observe the adhesion between the two cells under fluorescence microscope.Results:①Observed the growth characteristics under Inverted microscope, about 3～5 h, primary cultured cells start to adherent,48～72h, Cells entered the logarithmic phase, cytoplasm is rich and nucleu is clearly visible,5～7d, the cultured cells fuse and arrange monolayer "cobblestone " mosaic-like. Compared with the primary cells, the passaged cells grow vigorously, adherent early and have a morphologically similar.Ⅷfactor related antigen staining show that more than 96% endothelial cells are positive, CD34 positive rate is 89.38%, which illustrate that the isolated cells have a high purity and identify the cells as HUVECs;②The results of western blot and RT-PCR show that the expression of ERP is higher than the expression of ERαin HUVECs, the differences is statistically significant (t=-15.626, P<0.01; t=-3.995, P<0.05);③Comparison the adhesion between THP-1 and HUVECs gene transfected showed:the adhesion rate between THP-1 and HUVECs in ERαoverexpress group is significantly lower than the control group (Ve group), the difference is statistically significant (t=-47.117,P<0.01); after ERαknockout, the adhesion rate between THP-1 and HUVECs is significantly higher than the control Sc group (t=48.111, P<0.01), the adhesion rate in ERβknockout and overexpress groups have no significant difference than the control group, the differences are not statistically significant (t=2.64, P>0.05; t=-0.564, P>0.05)Conclution:①The technology of isolation in vitro, culture, cryopreservation, resuscitation and identification is mature and reliable, which provides an ideal methods and materials for the VEC experiments in vitro.②Estrogen Receptor mainly has two subtypes ERαand ERβ, which are widely distributed in the body and have a similar structure, but the dominant expression of ERαand ERβin different tissues is different, their actions are also different. This expriment shows that the expression of ERβis higher than the expression of ERa in HUVECs.③During the process of monocytes migrate and adhere to endothelial cells,the ovrexpression of ERαin HUVECs can significantly inhibit the adhsion between THP-1 and HUVECs, while ERβhasn't this action,which will provide a new theoretical basis and ideas for the clinical intervention and treatment of Atherosclerosis.
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