摘要
目的旨在优化分离原代人脐静脉内皮细胞(pHUVECs)技术,探索建立简便、高效和经济的沉默长链非编码RNA(lncRNA)的pHUVECs模型方法。方法实验所用脐带来自福建医科大学附属第一医院健康产妇剖宫产的新生儿脐带。使用胶原酶消化法分离pHUVECs,5%胎牛血清培养液[内皮细胞培养基(ECM)∶M199=1∶4]培养细胞。显微镜观察形态学及免疫荧光法检测pHUVECs特异性Ⅷ因子和CD31的表达。使用脂质体将不同浓度小干扰RNA(siRNA)-Fam转染pHUVECs,并设置CTL组、1 nmol/L siRNA-Fam组、10 nmol/L siRNA-Fam组、20 nmol/L siRNA-Fam组、50 nmol/L siRNA-Fam组、100 nmol/L siRNA-Fam组,倒置荧光显微镜观察各组荧光强度,筛选最佳siRNA转染浓度。设置CTL组、1μL RNAFIT组、3μL RNAFIT组、5μL RNAFIT组、10μL RNAFIT组,转染siRNA-SNHG8后,实时荧光定量逆转录聚合酶链式反应(RT-qPCR)检测SNHG8表达,筛选最佳RNAFIT转染浓度。设置CTL组、阴性对照组、si-SNHG81#组、si-SNHG82#组、si-SNHG83#组、si-SNHG81+2+3#组。脂质体转染法将siRNA导入pHUVECs,分别培养24、48和72 h。RT-qPCR检测lncRNA SNHG8的表达。结果pHUVECs在培养48 h后呈典型的鹅卵石样排列。免疫荧光结果显示,Ⅷ因子和CD31呈阳性。与CTL组比较,50 nmol/L siRNA-Fam组荧光强度最高,差异有统计学意义(t=32.020,P<0.0001),5和10μL RNAFIT组SNHG8表达均较低,差异均有统计学意义(t=36.030、19.890,P均<0.0001)。50nmol/L siRNA和5μL RNAFIT转染pHUVECs 24h后,RT-qPCR结果显示,与CTL组比较,si-SNHG83#组及si-SNHG81+2+3#组沉默SNHG8的效率最高,差异均有统计学意义(t=13.950、4.606,P均<0.05);转染48及72 h后,与CTL组比较,si-SNHG81+2+3#组沉默SNHG8的效率较高,差异均有统计学意义(t=48.620、24.160,P均<0.0001)。转染48及72 h后,与si-SNHG83#组比较,si-SNHG81+2+3#组沉默SNHG8的效率较高,差异均有统计学意义(t=9.316、4.055,P均<0.05)。同时si-SNHG81+2+3#组转染后呈时间依赖性抑制SNHG8表达,72 h组沉默效率最高,差异有统计学意义(t=24.160,P<0.0001)。结论本研究成功分离高纯度高活力pHUVECs,且将3条siRNA-SNHG8共同转染pHUVECs沉默效率最高。该方法简便、技术成熟、周期短、更经济,为今后lncRNA参与内皮相关疾病提供理想的体外模型。
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)∶M199=1∶4].The expression of pHUVECs⁃specific factorⅧand 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,1μL RNAFIT group,3μL RNAFIT group,5μL RNAFIT group,and 10μL 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⁃SNHG81#group,si⁃SNHG82#group,si⁃SNHG83#group and si⁃SNHG81+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 factorⅧand 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<0.0001),SNHG8 expression was lower in both 5 and 10μL RNAFIT groups,the differences were statistically significant(t=36.030,19.890;all P<0.0001).After transfecting pHUVECs with 50 nmol/L siRNA and 5μL RNAFIT for 24 h,RT⁃qPCR results showed that compared with CTL gorup,si⁃SNHG83#group and si⁃SNHG81+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⁃SNHG81+2+3#group had the highest efficiency of SNHG8 silencing,and the differences were statistically significant(t=48.620,24.160;all P<0.0001).And compared with the si⁃SNHG83#group,after transfection for 48 and 72 h,the si⁃SNHG81+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⁃SNHG81+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<0.0001).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.
作者
樊宗成
马康源
程小兵
陈鑫
林云钗
王来成
虞坚建
张顺鹏
彭峰
FAN Zongcheng;MA Kangyuan;CHENG Xiaobing;CHEN Xin;LIN Yunchai;WANG Laicheng;YU Jianjian;ZHANG Shunpeng;PENG Feng(Third Clinical College of Anhui Medical University,Hefei,Anhui 230032,China;Department of Cardiology,the Third People's Hospital of Hefei,Hefei,Anhui 230022,China;Department of Occupational Medicine,the Third People's Hospital of Hefei,Hefei,Anhui 230022,China;Department of Cardiology,the First Affiliated Hospital of Fujian Medical University,Fuzhou,Fujian 350005,China)
出处
《热带医学杂志》
CAS
2024年第8期1067-1072,1083,F0002,共8页
Journal of Tropical Medicine
基金
国家自然科学基金(81970370)
福建省科技创新联合基金(2021Y9121)
福建省卫生科技项目(2020QNA056)
福建省卫生健康科研人才培养项目(2019-1-40)
合肥市第三人民医院科研项目(SYKZ202201)。
关键词
原代人脐静脉内皮细胞
长链非编码RNA
脂质体转染
沉默效率
Primary human umbilical vein endothelial cells
Long non⁃coding RNA
Liposome transfection
Silencing efficiency
