中国危重病急救医学
中國危重病急救醫學
중국위중병급구의학
CHINESE CRITICAL CARE MEDICINE
2012年
9期
522-525
,共4页
高迁移率族蛋白B1%人脐静脉内皮细胞%转染%脓毒症%短发夹RNA
高遷移率族蛋白B1%人臍靜脈內皮細胞%轉染%膿毒癥%短髮夾RNA
고천이솔족단백B1%인제정맥내피세포%전염%농독증%단발협RNA
High mobility group box-1%Human umbilical vein endothelial cell%Transfection%Sepsis%Short hairpin RNA
目的 构建针对高迁移率族蛋白B1 (HMGB1)基因的特异性RNA干扰载体,建立稳定转染该干扰载体的人脐静脉内皮细胞株(HUVEC).方法 根据HMGB1基因的编码序列及短发夹RNA(shRNA)设计原则,设计并合成针对HMGB1基因的特异性shRNA,将其定向克隆到pRNA-u6.1/Neo载体中,同时设立阴性对照,重组载体经脂质体转染HUVEC;转染细胞经类氨基糖抗生素样物质G418筛选,采用实时荧光逆转录-聚合酶链反应检测HMGB1mRNA表达,蛋白质免疫印迹法检测HMGB1蛋白表达.结果 酶切鉴定和测序证实,重组质粒shRNA序列与设计的片段完全一致.HMGB1基因特异性RNA干扰载体能够显著抑制HMGB1在HUVEC细胞中的表达(mRNA:0.4635±0.0342比1.0340±0.0352,蛋白:0.4510 ± 0.0200比1.0210±0.0110,均P＜0.05).结论 成功构建了靶向HMGB1基因的shRNA真核表达载体pRNA-HMGB1,并获得了稳定表达靶向HMGB1基因的shRNA的HUVEC细胞株,为进一步研究HMGB1在HUVEC细胞株中的生物学功能和作用机制奠定了基础.
目的 構建針對高遷移率族蛋白B1 (HMGB1)基因的特異性RNA榦擾載體,建立穩定轉染該榦擾載體的人臍靜脈內皮細胞株(HUVEC).方法 根據HMGB1基因的編碼序列及短髮夾RNA(shRNA)設計原則,設計併閤成針對HMGB1基因的特異性shRNA,將其定嚮剋隆到pRNA-u6.1/Neo載體中,同時設立陰性對照,重組載體經脂質體轉染HUVEC;轉染細胞經類氨基糖抗生素樣物質G418篩選,採用實時熒光逆轉錄-聚閤酶鏈反應檢測HMGB1mRNA錶達,蛋白質免疫印跡法檢測HMGB1蛋白錶達.結果 酶切鑒定和測序證實,重組質粒shRNA序列與設計的片段完全一緻.HMGB1基因特異性RNA榦擾載體能夠顯著抑製HMGB1在HUVEC細胞中的錶達(mRNA:0.4635±0.0342比1.0340±0.0352,蛋白:0.4510 ± 0.0200比1.0210±0.0110,均P＜0.05).結論 成功構建瞭靶嚮HMGB1基因的shRNA真覈錶達載體pRNA-HMGB1,併穫得瞭穩定錶達靶嚮HMGB1基因的shRNA的HUVEC細胞株,為進一步研究HMGB1在HUVEC細胞株中的生物學功能和作用機製奠定瞭基礎.
목적 구건침대고천이솔족단백B1 (HMGB1)기인적특이성RNA간우재체,건립은정전염해간우재체적인제정맥내피세포주(HUVEC).방법 근거HMGB1기인적편마서렬급단발협RNA(shRNA)설계원칙,설계병합성침대HMGB1기인적특이성shRNA,장기정향극륭도pRNA-u6.1/Neo재체중,동시설립음성대조,중조재체경지질체전염HUVEC;전염세포경류안기당항생소양물질G418사선,채용실시형광역전록-취합매련반응검측HMGB1mRNA표체,단백질면역인적법검측HMGB1단백표체.결과 매절감정화측서증실,중조질립shRNA서렬여설계적편단완전일치.HMGB1기인특이성RNA간우재체능구현저억제HMGB1재HUVEC세포중적표체(mRNA:0.4635±0.0342비1.0340±0.0352,단백:0.4510 ± 0.0200비1.0210±0.0110,균P＜0.05).결론 성공구건료파향HMGB1기인적shRNA진핵표체재체pRNA-HMGB1,병획득료은정표체파향HMGB1기인적shRNA적HUVEC세포주,위진일보연구HMGB1재HUVEC세포주중적생물학공능화작용궤제전정료기출.
Objective To construct the short hairpin RNA (shRNA) targeting high mobility group box-1 (HMGB1) and culture the stable human umbilical vein endothelial cell (HUVEC) line expressing this shRNA.Methods Based on the HMGB1 gene sequence,shRNA was designed,synthesizedandsubclonedintothepRNA-u6.1/Neo vector,while negative controls were also established.Then the recombinant vector was transfected into HUVEC cell line and the cell was screened with G418 and assayed by using real time reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting.Results Restriction endonuclease digestion test and sequencing verification showed that the recombinant pRNA-u6.1/Neo vector expressing shRNA targeting HMGB1 was successfully constructed and the stable HUVEC cell line expressing this shRNA was developed.The real time RT-PCR and Western blotting was used to detect that recombinant plasmid in HUVEC cell effect on expression of HMGB1 was reduced (mRNA:0.4635 ± 0.0342 vs.1.0340 ± 0.0352,protein:0.45 10 ±0.0200 vs.1.0210 ±0.0110,both P＜0.05).Conclusion The recombinant pRNA-u6.1/Neo vector expressing shRNA targeting HMGB1 was successfully constructed and the stable HUVEC cell line expressing this shRNA was developed,and therefore allowed further investigation regarding the function of HMGB1 gene in the HUVEC cell line.
