Isolation of primary human umbilical vein endothelial cells and validation of the efficiency of silencing long non-coding RNA
Objective To optimize the isolation of primary human umbilical vein endothelial cells(pHUVECs),and explore a simple,efficient and economical pHUVECs model for silencing long non-coding RNA(lncRNA).Methods The umbilical cord used in the experiment was obtained from the neonatal umbilical cord of healthy puerpera in the First Affiliated Hospital of Fujian Medical University.The pHUVECs were isolated by collagenase digestion and cultured in 5％fetal bovine serum medium[endothelial cell medium(ECM):M 199=1:4].The expression of pHUVECs-specific factor Ⅷand CD31 was detected by microscopy and immunofluorescence.Different concentrations of small interfering RNA(siRNA)-Fam were transfected with pHUVECs using liposomes,CTL group,1 nmol/L siRNA-Fam group,10 nmol/L siRNA-Fam group,20 nmol/L siRNA-Fam group,50 nmol/L siRNA-Fam group and 100 nmol/L siRNA-Fam group were set up.The fluorescence intensity of each groups was observed by inverted fluorescence microscope,and the optimal siRNA transfection concentration was screened.CTL group,1 µL RNAFIT group,3 µL RNAFIT group,5 μL RNAFIT group,and 10 μL RNAFIT group were set up.After siRNA-SNHG8 transfection,reverse transcription-quantitative polymerase chain reaction(RT-qPCR)was used to detect SNHG8 expression and screen the optimal RNAFIT transfection concentration.CTL group,negative control group,si-SNHG8 1# group,si-SNHG8 2# group,si-SNHG8 3# group and si-SNHG8 1+2+3# group were set up for the studies.SiRNA was transfected into pHUVECs by liposome transfection and cultured for 24,48 and 72 h.The expression of lncRNA SNHG8 was detected by RT-qPCR.Results The pHUVECs showed a typical pebble-like arrangement after 48 h of culture.The factor Ⅷ and CD31 were positive by immunofluorescence assay in pHUVECs.Compared with CTL group,the fluorescence intensity of 50 nmol/L siRNA-Fam group was the highest,the difference was statistically significant(t=32.020,P＜0.000 1),SNHG8 expression was lower in both 5 and 10 μL RNAFIT groups,the differences were statistically significant(t=36.030,19.890;all P＜0.000 1).After transfecting pHUVECs with 50 nmol/L siRNA and 5 μL RNAFIT for 24 h,RT-qPCR results showed that compared with CTL gorup,si-SNHG8 3# group and si-SNHG8 1+2+3# group had the highest efficiency of SNHG8 silencing,and the differences were statistically significant(t=13.950,4.606;all P＜0.05).After transfection for 48 and 72 h,compared with CTL gorup,si-SNHG8 1+2+3# group had the highest efficiency of SNHG8 silencing,and the differences were statistically significant(t=48.620,24.160;allP＜0.000 1).And compared with the si-SNHG8 3# group,after transfection for 48 and 72 h,the si-SNHG8 1+2+3# group had a higher efficiency of silencing SNHG8,the differences were statistically significant(t=9.316,4.055;all P＜0.05).At the same time,the expression of SNHG8 in si-SNHG8 1+2+3# group was time-dependent after transfected,and the silence efficiency was the highest at 72 h,the difference was statistically significant(t=24.160,P＜0.000 1).Conclusions In this study,pHUVECs with high purity and high activity were isolated successfully,and the silencing efficiency of pHUVECs was the highest when three siRNA-SNHG8 were transfected together.This method was simple,mature,short and economical,and could provide an ideal in vitro model for the involvement of lncRNA in endothelium-related diseases in the future.
Primary human umbilical vein endothelial cellsLong non-coding RNALiposome transfectionSilencing efficiency
万方数据
维普
