国际麻醉学与复苏杂志
國際痳醉學與複囌雜誌
국제마취학여복소잡지
INTERNATIONAL JOURNAL OF ANESTHESIOLOGY AND RESUSCITATION
2013年
7期
595-599
,共5页
刘华跃%嵇富海%王秀云%左剑玲%许期年
劉華躍%嵇富海%王秀雲%左劍玲%許期年
류화약%혜부해%왕수운%좌검령%허기년
血管内皮细胞%小窝蛋白-1%小干扰RNA%磺基罗丹明B法%细胞毒性%筛选
血管內皮細胞%小窩蛋白-1%小榦擾RNA%磺基囉丹明B法%細胞毒性%篩選
혈관내피세포%소와단백-1%소간우RNA%광기라단명B법%세포독성%사선
Endothelial cells%Caveolin-1%Small interfering RNA%Sulforhodamine B%Cytotoxicity%Election
目的 筛选有效抑制血管内皮细胞小窝蛋白-1(Caveolin-1)表达的小干扰RNA(small interferenceRNA,siRNA)序列,并检测转染复合物的细胞毒性. 方法 设计合成针对Caveolin-1的siRNA 3条(siRNA422,siRNA548,siRNA710)及1条带绿色荧光标记的通用阴性对照FAM-siRNA.在siRNAFect介导下转染血管内皮细胞.用实时荧光定量PCR(Real-time PCR)测定转染后血管内皮细胞中Caveolin-1 mRNA的表达,Western blot方法测定干扰后Caveolin-1蛋白表达,比较抑制率,筛选出有效抑制Caveolin-1表达的siRNA.采用磺基罗丹明B法(sulforhodamineB,SRB)分析转染复合物的细胞毒性. 结果 ①在siRNAFect相同剂量下,siRNA 20、30 nmol/L组或40 nmol/L组转染效率均达到85％以上(P＜0.01)；②siRNAFect 1.3μl复合10 nmol/L或20 nmol/L siRNA组细胞存活率均达到80％以上(P＜0.05)；③siRNA548对EA.hy926细胞Caveolin-1基因mRNA抑制效果最明显(P＜0.01)；④与阴性对照组及siRNA422、siRNA710相比较,siRNA548干扰血管内皮细胞48 h后,Caveolin-1蛋白的表达量最低(P＜0.01). 结论 ①siRNA548对血管内皮细胞Caveolin-1表达的抑制效果最大.②siRNA浓度20 nmol/L复合siRNAFect 1.3μl转染效率高,细胞毒性低,适合转染.
目的 篩選有效抑製血管內皮細胞小窩蛋白-1(Caveolin-1)錶達的小榦擾RNA(small interferenceRNA,siRNA)序列,併檢測轉染複閤物的細胞毒性. 方法 設計閤成針對Caveolin-1的siRNA 3條(siRNA422,siRNA548,siRNA710)及1條帶綠色熒光標記的通用陰性對照FAM-siRNA.在siRNAFect介導下轉染血管內皮細胞.用實時熒光定量PCR(Real-time PCR)測定轉染後血管內皮細胞中Caveolin-1 mRNA的錶達,Western blot方法測定榦擾後Caveolin-1蛋白錶達,比較抑製率,篩選齣有效抑製Caveolin-1錶達的siRNA.採用磺基囉丹明B法(sulforhodamineB,SRB)分析轉染複閤物的細胞毒性. 結果 ①在siRNAFect相同劑量下,siRNA 20、30 nmol/L組或40 nmol/L組轉染效率均達到85％以上(P＜0.01)；②siRNAFect 1.3μl複閤10 nmol/L或20 nmol/L siRNA組細胞存活率均達到80％以上(P＜0.05)；③siRNA548對EA.hy926細胞Caveolin-1基因mRNA抑製效果最明顯(P＜0.01)；④與陰性對照組及siRNA422、siRNA710相比較,siRNA548榦擾血管內皮細胞48 h後,Caveolin-1蛋白的錶達量最低(P＜0.01). 結論 ①siRNA548對血管內皮細胞Caveolin-1錶達的抑製效果最大.②siRNA濃度20 nmol/L複閤siRNAFect 1.3μl轉染效率高,細胞毒性低,適閤轉染.
목적 사선유효억제혈관내피세포소와단백-1(Caveolin-1)표체적소간우RNA(small interferenceRNA,siRNA)서렬,병검측전염복합물적세포독성. 방법 설계합성침대Caveolin-1적siRNA 3조(siRNA422,siRNA548,siRNA710)급1조대록색형광표기적통용음성대조FAM-siRNA.재siRNAFect개도하전염혈관내피세포.용실시형광정량PCR(Real-time PCR)측정전염후혈관내피세포중Caveolin-1 mRNA적표체,Western blot방법측정간우후Caveolin-1단백표체,비교억제솔,사선출유효억제Caveolin-1표체적siRNA.채용광기라단명B법(sulforhodamineB,SRB)분석전염복합물적세포독성. 결과 ①재siRNAFect상동제량하,siRNA 20、30 nmol/L조혹40 nmol/L조전염효솔균체도85％이상(P＜0.01)；②siRNAFect 1.3μl복합10 nmol/L혹20 nmol/L siRNA조세포존활솔균체도80％이상(P＜0.05)；③siRNA548대EA.hy926세포Caveolin-1기인mRNA억제효과최명현(P＜0.01)；④여음성대조조급siRNA422、siRNA710상비교,siRNA548간우혈관내피세포48 h후,Caveolin-1단백적표체량최저(P＜0.01). 결론 ①siRNA548대혈관내피세포Caveolin-1표체적억제효과최대.②siRNA농도20 nmol/L복합siRNAFect 1.3μl전염효솔고,세포독성저,괄합전염.
Objective Aim to pick out the small interference RNA (siRNA) which could most effectively inhibit the expression of caveolin-1 in endothelial cells and to detect the cytotoxicity of the transfection complex.Methods Four siRNAs were chemically synthesized:siRNA422,siRNA548,siRNA710 were used to inhibit caveolin-1 expression in endothelial cells,FAM-labeled mismatch siRNA was as a negative control They were all transfected into endothelial cells,respectively.Caveolin-1 mRNA was detected after transfection by real-time PCR and its protein expression was measured by Western blot.The cytotoxicity was analyzed with the method of the sulforhodamine B (SRB).Results ① The transfecfon rates are all above 85％ under the transfection condition of siRNA(20,30 nmol/L or 40 nmol/L) combined with the same dose of siRNAFect(P＜0.01).② The survival rates of transfected cells are all above 80％ under the transfection condition of siRNAFect 1.3 μl combined with siRNA (10 nmol/L or 20 nmol/L)(P＜0.05).③ The expression of caveolin-1 mRNA in EA.hy926 cells was significantly decreased after siRNA548 treatment (P＜0.01).④ Compared with siRNA422,siRNA710,vehicle or mismatch siRNA-treated group,the expression of caveolin-1 protein was the least after siRNA548 treatment 48 h later(P＜0.01).Conclusions ① The siRNA548 can inhibit caveolin-1 expression most effectively in endothelial cells.② siRNA(20 nmol/L) combined with siRNAFect(1.3 μl) has lower cytotoxicity and higher transfection efficiency,which is suitable for transfection.
