中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2008年
7期
881-883,封4
,共4页
王海波%刘相萍%杨堃%隋爱华%刘春远%周岩冰
王海波%劉相萍%楊堃%隋愛華%劉春遠%週巖冰
왕해파%류상평%양곤%수애화%류춘원%주암빙
真核表达载体%胃癌%基因表达
真覈錶達載體%胃癌%基因錶達
진핵표체재체%위암%기인표체
Eukaryotic double expression vector%Gastric carcinoma%Gene expression
目的 构建p21WAF1与p27KIP1真核双表达载体,体外转染胃癌7901细胞,观察其在胃癌细胞中的表达及相关特性.方法 提取人全血总RNA,利用逆转录-聚合酶链反应(RT-PCR)扩增p21WAF1和p27WAF1基因并将其分别克隆到真核双表达载体pIRES中构建重组质粒pIPES-p27KIP1-p21WAF1,经酶切和测序鉴定重组质粒.脂质体介导重组质粒plRES-p27KIP1.p21WAF1"转染胃癌7901细胞,收集并提取转染后12、24、48、72 h和5 d各细胞的总RNA,逆转录成cDNA,荧光定量聚合酶链反应(FQ-PCR)检测各细胞中p21WAF1和p27KIP1在RNA水平的表达.带有绿色荧光蛋白的pIRES-EGFP作为对照.结果 RT-PCR扩增分别获得约500和600 bp大小的产物,重组质粒pIRES-p27KIP1.p21WAF1经酶切和测序鉴定证实为p21WAF1和p27KIP1基因.转染胃癌7901细胞48 h时转染率为68%.与空质粒对照组比较,重组质粒转染胃癌7901细胞12、24、48、72 h和5 d后FQ-PCR证实p27KIP1的Ct值(cycle threshold,Ct)分别减少8.2、10、10、7.4和6.7,p21WAF1的Ct值分别减少7.4、8.2、8.2、6.3和5.70其中24 h和48 h的Ct值减少最大.结论 真核双表达载体pIRES-p27KIP1.p21WAF1构建成功并能在胃癌7901细胞内高效表达.
目的 構建p21WAF1與p27KIP1真覈雙錶達載體,體外轉染胃癌7901細胞,觀察其在胃癌細胞中的錶達及相關特性.方法 提取人全血總RNA,利用逆轉錄-聚閤酶鏈反應(RT-PCR)擴增p21WAF1和p27WAF1基因併將其分彆剋隆到真覈雙錶達載體pIRES中構建重組質粒pIPES-p27KIP1-p21WAF1,經酶切和測序鑒定重組質粒.脂質體介導重組質粒plRES-p27KIP1.p21WAF1"轉染胃癌7901細胞,收集併提取轉染後12、24、48、72 h和5 d各細胞的總RNA,逆轉錄成cDNA,熒光定量聚閤酶鏈反應(FQ-PCR)檢測各細胞中p21WAF1和p27KIP1在RNA水平的錶達.帶有綠色熒光蛋白的pIRES-EGFP作為對照.結果 RT-PCR擴增分彆穫得約500和600 bp大小的產物,重組質粒pIRES-p27KIP1.p21WAF1經酶切和測序鑒定證實為p21WAF1和p27KIP1基因.轉染胃癌7901細胞48 h時轉染率為68%.與空質粒對照組比較,重組質粒轉染胃癌7901細胞12、24、48、72 h和5 d後FQ-PCR證實p27KIP1的Ct值(cycle threshold,Ct)分彆減少8.2、10、10、7.4和6.7,p21WAF1的Ct值分彆減少7.4、8.2、8.2、6.3和5.70其中24 h和48 h的Ct值減少最大.結論 真覈雙錶達載體pIRES-p27KIP1.p21WAF1構建成功併能在胃癌7901細胞內高效錶達.
목적 구건p21WAF1여p27KIP1진핵쌍표체재체,체외전염위암7901세포,관찰기재위암세포중적표체급상관특성.방법 제취인전혈총RNA,이용역전록-취합매련반응(RT-PCR)확증p21WAF1화p27WAF1기인병장기분별극륭도진핵쌍표체재체pIRES중구건중조질립pIPES-p27KIP1-p21WAF1,경매절화측서감정중조질립.지질체개도중조질립plRES-p27KIP1.p21WAF1"전염위암7901세포,수집병제취전염후12、24、48、72 h화5 d각세포적총RNA,역전록성cDNA,형광정량취합매련반응(FQ-PCR)검측각세포중p21WAF1화p27KIP1재RNA수평적표체.대유록색형광단백적pIRES-EGFP작위대조.결과 RT-PCR확증분별획득약500화600 bp대소적산물,중조질립pIRES-p27KIP1.p21WAF1경매절화측서감정증실위p21WAF1화p27KIP1기인.전염위암7901세포48 h시전염솔위68%.여공질립대조조비교,중조질립전염위암7901세포12、24、48、72 h화5 d후FQ-PCR증실p27KIP1적Ct치(cycle threshold,Ct)분별감소8.2、10、10、7.4화6.7,p21WAF1적Ct치분별감소7.4、8.2、8.2、6.3화5.70기중24 h화48 h적Ct치감소최대.결론 진핵쌍표체재체pIRES-p27KIP1.p21WAF1구건성공병능재위암7901세포내고효표체.
Objective To construct and identify recombinant eukaryotie double expression vector pIRES-p27KIP1-p21WAF1 and observe the expression of the recombinant plRES-p27KIP1-p21WAF1 in human gastric carcinoma cells SGC-7901. Methods Total RNA was isolated from healthy human blood. The full-length open reading frames of human p21WAF1 and p27KIP1 gene were amplified by RT-PCR and cloned into eukaryotic double expression plasmid pIRES. After identified by enzyme digestion and DNA sequencing, the recombinant plasmid was transfected into human gastric carcinoma cells SGC-7901 with liposomes. The fluorescence quantitative polymerase chain reaction (FQ-PCR) was used to detect the mRNA expression. The pIRES-EGFP plasmid with green fluorescence protein served as control. Results The recombinant plasmid was constructed successfully,which was confirmed by enzyme digestion and DNA sequencing. The transfection rate of human gastric carcinoma cells was 68% 48 h after transfection. Compared with blank vector group,12,24,48,72 h and 5 days after transfection with plRES-p27KIP1-p21WAF1 ,the Ct value of p27KIP1 was reduced by 8.2,10,10,7.4 and 6.7, and that of p21WAF1 was reduced by 7.4,8.2,8.2,6.3and 5.7 respectively. Conclusion The recombinant pIRES-p27KIP1-p21WAF1 was constructed successfully and expressed effectively in human gastric carcinoma cells.
