中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2013年
12期
711-714
,共4页
人脐静脉内皮细胞%高迁移率族蛋白B1%增殖%迁移
人臍靜脈內皮細胞%高遷移率族蛋白B1%增殖%遷移
인제정맥내피세포%고천이솔족단백B1%증식%천이
Human umbilical vein endothelial cell%High mobility group box-1%Proliferation%Migration
目的 探讨高迁移率族蛋白B1 (HMGB1)对人脐静脉内皮细胞(HUVEC)增殖和迁移能力的影响.方法 通过稳定转染的方法将pRNA-u6.1/Neo-对照和pRNA-u6.1/Neo-HMGB1短发夹RNA(shRNA)质粒转至HUVEC细胞,构建对照及HMGB1 shRNA细胞株,通过蛋白质免疫印迹试验(Western blotting)和实时荧光逆转录-聚合酶链反应(RT-PCR)进行稳定转染细胞的鉴定,通过四甲基偶氮唑盐(MTT)检测细胞株增殖情况,流式细胞仪检测细胞株周期的分布,划痕实验检测细胞株迁移能力.结果 Western blotting及RT-PCR检测证明HMGB1 shRNA稳定转染的HUVEC细胞株构建成功,培养72 h时细胞中HMGB1蛋白及mRNA表达较对照细胞明显降低(蛋白:0.436±0.027比1.017±0.038,T=12.180,P=0.000; mRNA:0.436±0.031比1.020±0.051,T=9.660,P=0.001).MTT结果显示,HMGB1 shRNA稳定转染HUVEC细胞株2、3、4、5d的增殖能力比对照细胞株明显下降(2 d:0.210±0.023比0.240±0.011,T=1.050,P=0.351;3 d:0.240±0.022比0.361±0.030,T=3.203,P=0.033;4 d:0.373±0.031比0.531±0.033,T=3.530,P=0.022;5 d:0.441±0.031比0.602±0.030,T=4.180,P=0.106).流式细胞仪检测结果显示,HMGB1 shRNA细胞株在S期的比例明显低于对照细胞株[(13.10±1.10)％比(21.12±1.20)％,T=4.950,P=0.001].划痕实验结果显示,HMGB1 shRNA细胞株在细胞刮除后12h划痕愈合的细胞比例较对照细胞株明显降低[(21.07±3.33)％比(88.53±3.15)％,T=14.142,P=0.000].结论 HMGB1 shRNA能明显抑制HUVEC细胞的增殖和迁移能力.
目的 探討高遷移率族蛋白B1 (HMGB1)對人臍靜脈內皮細胞(HUVEC)增殖和遷移能力的影響.方法 通過穩定轉染的方法將pRNA-u6.1/Neo-對照和pRNA-u6.1/Neo-HMGB1短髮夾RNA(shRNA)質粒轉至HUVEC細胞,構建對照及HMGB1 shRNA細胞株,通過蛋白質免疫印跡試驗(Western blotting)和實時熒光逆轉錄-聚閤酶鏈反應(RT-PCR)進行穩定轉染細胞的鑒定,通過四甲基偶氮唑鹽(MTT)檢測細胞株增殖情況,流式細胞儀檢測細胞株週期的分佈,劃痕實驗檢測細胞株遷移能力.結果 Western blotting及RT-PCR檢測證明HMGB1 shRNA穩定轉染的HUVEC細胞株構建成功,培養72 h時細胞中HMGB1蛋白及mRNA錶達較對照細胞明顯降低(蛋白:0.436±0.027比1.017±0.038,T=12.180,P=0.000; mRNA:0.436±0.031比1.020±0.051,T=9.660,P=0.001).MTT結果顯示,HMGB1 shRNA穩定轉染HUVEC細胞株2、3、4、5d的增殖能力比對照細胞株明顯下降(2 d:0.210±0.023比0.240±0.011,T=1.050,P=0.351;3 d:0.240±0.022比0.361±0.030,T=3.203,P=0.033;4 d:0.373±0.031比0.531±0.033,T=3.530,P=0.022;5 d:0.441±0.031比0.602±0.030,T=4.180,P=0.106).流式細胞儀檢測結果顯示,HMGB1 shRNA細胞株在S期的比例明顯低于對照細胞株[(13.10±1.10)％比(21.12±1.20)％,T=4.950,P=0.001].劃痕實驗結果顯示,HMGB1 shRNA細胞株在細胞颳除後12h劃痕愈閤的細胞比例較對照細胞株明顯降低[(21.07±3.33)％比(88.53±3.15)％,T=14.142,P=0.000].結論 HMGB1 shRNA能明顯抑製HUVEC細胞的增殖和遷移能力.
목적 탐토고천이솔족단백B1 (HMGB1)대인제정맥내피세포(HUVEC)증식화천이능력적영향.방법 통과은정전염적방법장pRNA-u6.1/Neo-대조화pRNA-u6.1/Neo-HMGB1단발협RNA(shRNA)질립전지HUVEC세포,구건대조급HMGB1 shRNA세포주,통과단백질면역인적시험(Western blotting)화실시형광역전록-취합매련반응(RT-PCR)진행은정전염세포적감정,통과사갑기우담서염(MTT)검측세포주증식정황,류식세포의검측세포주주기적분포,화흔실험검측세포주천이능력.결과 Western blotting급RT-PCR검측증명HMGB1 shRNA은정전염적HUVEC세포주구건성공,배양72 h시세포중HMGB1단백급mRNA표체교대조세포명현강저(단백:0.436±0.027비1.017±0.038,T=12.180,P=0.000; mRNA:0.436±0.031비1.020±0.051,T=9.660,P=0.001).MTT결과현시,HMGB1 shRNA은정전염HUVEC세포주2、3、4、5d적증식능력비대조세포주명현하강(2 d:0.210±0.023비0.240±0.011,T=1.050,P=0.351;3 d:0.240±0.022비0.361±0.030,T=3.203,P=0.033;4 d:0.373±0.031비0.531±0.033,T=3.530,P=0.022;5 d:0.441±0.031비0.602±0.030,T=4.180,P=0.106).류식세포의검측결과현시,HMGB1 shRNA세포주재S기적비례명현저우대조세포주[(13.10±1.10)％비(21.12±1.20)％,T=4.950,P=0.001].화흔실험결과현시,HMGB1 shRNA세포주재세포괄제후12h화흔유합적세포비례교대조세포주명현강저[(21.07±3.33)％비(88.53±3.15)％,T=14.142,P=0.000].결론 HMGB1 shRNA능명현억제HUVEC세포적증식화천이능력.
Objective To investigate the effect of down-regulation of high mobility group box-1 (HMGB1) expression on the proliferation and migration abilities of human umbilical vein endothelial cell (HUVEC) in vitro.Methods The method of stable transfection was used to transfect the pRNA-u6.1/Neo-control and pRNA-u6.1/ Neo-HMGB1 short hairpin RNA (shRNA) plasmid to HUVEC cells,and control and HMGB1 shRNA cell lines were reproduced,and the cells were identified by Western blotting test and real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR).Then methyl thiazolyl tetrazolium (MTT) was used for the determination of the proliferative ability,flow cytometry was used for determining the changes in cell cycle distribution ability of HUVEC,and the migratory ability was detected by scratch test.Results Western blotting and RT-PCR detection proved the production of stable transfection cell lines was successful,and the content of HMGB1 protein and mRNA expression in cells were significantly lower after cultivation for 72 hours than those of control cells (protein:0.436 ±0.027 vs.1.017 ±0.038,_T=12.180,P=0.000; mRNA:0.436 ±0.031 vs.1.020±0.051,T=9.660,P=0.001).The results of MTT showed that the proliferation ability of HMGB1 shRNA cell lines was lower obviously 2,3,4,5 days after transfection than that of control cells (2 days:0.210 ± 0.023 vs.0.240 ± 0.011,T=1.050,P=0.351 ; 3 days:0.240 ± 0.022 vs.0.361 ± 0.030,T=3.203,P=0.033; 4 days:0.373 ± 0.031 vs.0.531 ± 0.033,T=3.530,P=0.022; 5 days:0.441 ±0.031 vs.0.602 ±0.030,T=4.180,P=0.106).Flow cytometry results showed that the number of HMGB 1 shRNA cells in S phase was significantly lower than that of the control cell line [(13.10 ± 1.10)％ vs.(21.12 ± 1.20)％,T=4.950,P=0.001].Scratch test showed that the healing ability of HMGB1 shRNA cell line was lowered significantly at 12 hours as compared with that of control [(20.17 ± 3.33)％ vs.(88.53 ± 3.15)％,T=14.142,P=0.000].Conclusion HMGB1 shRNA can significantly inhibit HUVEC cell proliferation and migration.
