摘要
目的：探讨高迁移率族蛋白B1（HMGB1）基因表达对人脐静脉血管内皮细胞（HUVEC）激活表达E-选择素的分子机制。方法将同源盒转录因子（HOXA9）小干扰RNA（siRNA）短序列转染至对数生长期的HUVEC，采用实时荧光定量聚合酶链反应（实时qPCR）和蛋白质免疫印迹试验（Western Blot）检测其对HOXA9 mRNA和蛋白表达的影响；另设空白对照组和无意序列nonsilence阴性对照组。取已经稳定转染pRNA-u6.1/Neo-HMGB1 shRNA质粒的HUVEC（低表达HMGB1的HUVEC），采用实时qPCR法检测HOXA9和E选择素的mRNA表达；另设nonsilence转染组作为阴性对照。将HOXA9 siRNA转染至低表达HMGB1的HUVEC中作为共同转染组，采用实时qPCR法检测E-选择素的mRNA表达；以HMGB1 shRNA组和HOXA9 nonsilence组作为对照。结果①空白对照组HOXA9 mRNA（2-ΔΔCT）和蛋白（积分A值）表达分别为1.094±0.115和1.031±0.060。与无意序列nonsilence转染组比较， HOXA9 siRNA转染组可显著降低HUVEC细胞中HOXA9的mRNA和蛋白表达〔HOXA9 mRNA（2-ΔΔCT）：0.257±0.030比1.035±0.091，t＝14.010，P＝0.002；HOXA9蛋白（积分A值）：0.278±0.042比0.975±0.014，t＝27.310，P＝0.002〕。②与nonsilence转染组比较， HMGB1 shRNA转染上调了HUVEC中HOXA9 mRNA（2-ΔΔCT）表达（2.519±0.278比0.856±0.063，t＝10.100， P＝0.001），同时下调了E-选择素mRNA（2-ΔΔCT）表达（0.311±0.046比1.080±0.201，t＝7.415，P＝0.000）。③与HOXA9 nonsilence组及HMGB1 shRNA组比较，HMGB1 shRNA和HOXA9 siRNA共同转染后HUVEC细胞中E-选择素 mRNA（2-ΔΔCT）表达明显升高（3.445±0.428比1.085±0.212、1.004±0.104，t1＝8.507， t2＝9.603，均P＜0.001）。结论 HMGB1在HUVEC细胞核内可能通过HOXA9调节E-选择素的表达。
ObjectiveTo approach the regulatory mechanism of high mobility group box-1 （HMGB1） on the expression of E-selectin in human umbilical vein endothelial cell （HUVEC）.Methods Homeobox A9 （HOXA9） siRNA was transfected to HUVEC at logarithmic phase, real-time fluorescence quantitative polymerase chain reaction （real-time qPCR） and Western Blot were used to determine the HOXA9 mRNA expression and protein expressions; a blank control group and a nonsilence negative control group were set. HUVEC stable transfected with pRNA-u6.1/Neo-HMGB1 shRNA plasmids （HUVEC with low-expression HMGB1） was obtained, and HOXA9 and E-selectin mRNA expressions were determined with real-time qPCR; a nonsilence transfection group served as the negative control. The HOXA9 siRNA was transfected to HUVEC with low-expression HMGB1 as co-transfection group, and the E-selectin expressions was determined with real-time qPCR; a HMGB1 shRNA group and a HOXA9 nonsilence group served as control.Results① HOXA9 mRNA （2-ΔΔCT） and protein expression （integralA value） in blank control group were 1.094±0.115 and 1.031±0.060. Compared with nonsilence transfection group, HOXA9 siRNA transfection group could significantly reduced mRNA and protein expression of HOXA9 [HOXA9 mRNA （2-ΔΔCT）： 0.257±0.030 vs. 1.035±0.091,t = 14.010,P = 0.002; HOXA9 protein （integralA value）： 0.278±0.042 vs. 0.975±0.014,t = 27.310, P = 0.002].② Compared with nonsilence transfection group, HMGB1 shRNA transfection could up-regulate HOXA9 mRNA expression in HUVEC （2-ΔΔCT： 2.519±0.278 vs. 0.856±0.063,t = 10.100,P = 0.001）, also could down-regulate E-selectin mRNA expression （0.311±0.046 vs. 1.080±0.201,t = 7.415,P = 0.000）.③ Compared with HOXA9 nonsilence group and HMGB1 shRNA group, HMGB1 shRNA and HOXA9 siRNA co-transfected HUVEC cells could significantly elevate E-selectin mRNA expression （2-ΔΔCT： 3.445±0.428 vs. 1.085±0.212, 1.004±0.104,t1 = 8.507, t2 = 9.603, bothP〈 0.001）.Conclusion HMGB1 may regul
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2015年第8期662-666,共5页
Chinese Critical Care Medicine
基金
国家自然科学基金（30901438,81301619）
辽宁省沈阳市科技计划项目（F13-220-9-11）
