中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2015年
8期
662-666
,共5页
张晓娟%焦丽丽%栾正刚%马晓春
張曉娟%焦麗麗%欒正剛%馬曉春
장효연%초려려%란정강%마효춘
脓毒症%血管内皮细胞%高迁移率族蛋白%同源盒蛋白基因%E-选择素%转染%真核细胞表达载体
膿毒癥%血管內皮細胞%高遷移率族蛋白%同源盒蛋白基因%E-選擇素%轉染%真覈細胞錶達載體
농독증%혈관내피세포%고천이솔족단백%동원합단백기인%E-선택소%전염%진핵세포표체재체
Sepsis%Vascular endothelial cell%High mobility group protein%Homeobox gene%E-selectin%Transfection%Eukaryotic expression vector
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。
目的：探討高遷移率族蛋白B1（HMGB1）基因錶達對人臍靜脈血管內皮細胞（HUVEC）激活錶達E-選擇素的分子機製。方法將同源盒轉錄因子（HOXA9）小榦擾RNA（siRNA）短序列轉染至對數生長期的HUVEC，採用實時熒光定量聚閤酶鏈反應（實時qPCR）和蛋白質免疫印跡試驗（Western Blot）檢測其對HOXA9 mRNA和蛋白錶達的影響；另設空白對照組和無意序列nonsilence陰性對照組。取已經穩定轉染pRNA-u6.1/Neo-HMGB1 shRNA質粒的HUVEC（低錶達HMGB1的HUVEC），採用實時qPCR法檢測HOXA9和E選擇素的mRNA錶達；另設nonsilence轉染組作為陰性對照。將HOXA9 siRNA轉染至低錶達HMGB1的HUVEC中作為共同轉染組，採用實時qPCR法檢測E-選擇素的mRNA錶達；以HMGB1 shRNA組和HOXA9 nonsilence組作為對照。結果①空白對照組HOXA9 mRNA（2-ΔΔCT）和蛋白（積分A值）錶達分彆為1.094±0.115和1.031±0.060。與無意序列nonsilence轉染組比較， HOXA9 siRNA轉染組可顯著降低HUVEC細胞中HOXA9的mRNA和蛋白錶達〔HOXA9 mRNA（2-ΔΔCT）：0.257±0.030比1.035±0.091，t＝14.010，P＝0.002；HOXA9蛋白（積分A值）：0.278±0.042比0.975±0.014，t＝27.310，P＝0.002〕。②與nonsilence轉染組比較， HMGB1 shRNA轉染上調瞭HUVEC中HOXA9 mRNA（2-ΔΔCT）錶達（2.519±0.278比0.856±0.063，t＝10.100， P＝0.001），同時下調瞭E-選擇素mRNA（2-ΔΔCT）錶達（0.311±0.046比1.080±0.201，t＝7.415，P＝0.000）。③與HOXA9 nonsilence組及HMGB1 shRNA組比較，HMGB1 shRNA和HOXA9 siRNA共同轉染後HUVEC細胞中E-選擇素 mRNA（2-ΔΔCT）錶達明顯升高（3.445±0.428比1.085±0.212、1.004±0.104，t1＝8.507， t2＝9.603，均P＜0.001）。結論 HMGB1在HUVEC細胞覈內可能通過HOXA9調節E-選擇素的錶達。
목적：탐토고천이솔족단백B1（HMGB1）기인표체대인제정맥혈관내피세포（HUVEC）격활표체E-선택소적분자궤제。방법장동원합전록인자（HOXA9）소간우RNA（siRNA）단서렬전염지대수생장기적HUVEC，채용실시형광정량취합매련반응（실시qPCR）화단백질면역인적시험（Western Blot）검측기대HOXA9 mRNA화단백표체적영향；령설공백대조조화무의서렬nonsilence음성대조조。취이경은정전염pRNA-u6.1/Neo-HMGB1 shRNA질립적HUVEC（저표체HMGB1적HUVEC），채용실시qPCR법검측HOXA9화E선택소적mRNA표체；령설nonsilence전염조작위음성대조。장HOXA9 siRNA전염지저표체HMGB1적HUVEC중작위공동전염조，채용실시qPCR법검측E-선택소적mRNA표체；이HMGB1 shRNA조화HOXA9 nonsilence조작위대조。결과①공백대조조HOXA9 mRNA（2-ΔΔCT）화단백（적분A치）표체분별위1.094±0.115화1.031±0.060。여무의서렬nonsilence전염조비교， HOXA9 siRNA전염조가현저강저HUVEC세포중HOXA9적mRNA화단백표체〔HOXA9 mRNA（2-ΔΔCT）：0.257±0.030비1.035±0.091，t＝14.010，P＝0.002；HOXA9단백（적분A치）：0.278±0.042비0.975±0.014，t＝27.310，P＝0.002〕。②여nonsilence전염조비교， HMGB1 shRNA전염상조료HUVEC중HOXA9 mRNA（2-ΔΔCT）표체（2.519±0.278비0.856±0.063，t＝10.100， P＝0.001），동시하조료E-선택소mRNA（2-ΔΔCT）표체（0.311±0.046비1.080±0.201，t＝7.415，P＝0.000）。③여HOXA9 nonsilence조급HMGB1 shRNA조비교，HMGB1 shRNA화HOXA9 siRNA공동전염후HUVEC세포중E-선택소 mRNA（2-ΔΔCT）표체명현승고（3.445±0.428비1.085±0.212、1.004±0.104，t1＝8.507， t2＝9.603，균P＜0.001）。결론 HMGB1재HUVEC세포핵내가능통과HOXA9조절E-선택소적표체。
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 regulate E-selectin expression through the HOXA9 regulation in HUVEC.
