中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2009年
7期
880-883
,共4页
张靖%付彦超%康春生%张庆瑜
張靖%付彥超%康春生%張慶瑜
장정%부언초%강춘생%장경유
胃癌%蛋白激酶B1%环氧合酶2%短发夹RNA%腺病毒载体
胃癌%蛋白激酶B1%環氧閤酶2%短髮夾RNA%腺病毒載體
위암%단백격매B1%배양합매2%단발협RNA%선병독재체
Gastric carcinoma%Protein kinase B1%Cyclooxygenase 2%Short hairpin RNA%Adenovirus vector
目的 构建靶向性蛋白激酶B1(PKB1/Akt1)和环氧合酶-2(COX-2)的短发夹RNA(shRNA)腺病毒载体,观察其在人胃癌细胞株SGC-7901中的表达.方法 利用同源重组技术构建重组腺病毒载体pGSadeno-Akt1+COX-2(pGSadeno-A+C),经酶切及测序鉴定后转染人胚肾细胞HEK293包装成为重组rAd5-A+C腺病毒,并测定病毒的滴度.体外转染人胃癌细胞株SGC-7901后,Real-time PCR和蛋白印迹分别检测Akt1和COX-2 mRNA和蛋白质的表达.结果 成功构建rAd5-A+C重组腺病毒,测定包装的病毒滴度为1.0×1010pfu/ml.转染组Akt1和COX-2 mRNA表达明显下调,其△Ct值分别是(12.26±0.05)和(5.41±0.09),比rAd5-HK空载组(10.63±0.02)、(3.75±0.08)和对照组(10.57±0.02)、(3.73±0.08)增高(P<0.01),而空载组和对照组比较ΔCt值无明显变化(P>0.05).同样转染组Akt1和COX-2蛋白表达量与空载组和对照组比较分别下调70.5%和63.7%(P<0.01),空载组和对照组比较Akt1和COX-2蛋白表达差异无统计学意义(P>0.05).结论 靶向性Akt1和COX-2的shRNA腺病毒载体可以特异性抑制Akt1和COX-2的表达,可能成为胃癌靶向性Akt1和COX-2基因治疗的新策略.
目的 構建靶嚮性蛋白激酶B1(PKB1/Akt1)和環氧閤酶-2(COX-2)的短髮夾RNA(shRNA)腺病毒載體,觀察其在人胃癌細胞株SGC-7901中的錶達.方法 利用同源重組技術構建重組腺病毒載體pGSadeno-Akt1+COX-2(pGSadeno-A+C),經酶切及測序鑒定後轉染人胚腎細胞HEK293包裝成為重組rAd5-A+C腺病毒,併測定病毒的滴度.體外轉染人胃癌細胞株SGC-7901後,Real-time PCR和蛋白印跡分彆檢測Akt1和COX-2 mRNA和蛋白質的錶達.結果 成功構建rAd5-A+C重組腺病毒,測定包裝的病毒滴度為1.0×1010pfu/ml.轉染組Akt1和COX-2 mRNA錶達明顯下調,其△Ct值分彆是(12.26±0.05)和(5.41±0.09),比rAd5-HK空載組(10.63±0.02)、(3.75±0.08)和對照組(10.57±0.02)、(3.73±0.08)增高(P<0.01),而空載組和對照組比較ΔCt值無明顯變化(P>0.05).同樣轉染組Akt1和COX-2蛋白錶達量與空載組和對照組比較分彆下調70.5%和63.7%(P<0.01),空載組和對照組比較Akt1和COX-2蛋白錶達差異無統計學意義(P>0.05).結論 靶嚮性Akt1和COX-2的shRNA腺病毒載體可以特異性抑製Akt1和COX-2的錶達,可能成為胃癌靶嚮性Akt1和COX-2基因治療的新策略.
목적 구건파향성단백격매B1(PKB1/Akt1)화배양합매-2(COX-2)적단발협RNA(shRNA)선병독재체,관찰기재인위암세포주SGC-7901중적표체.방법 이용동원중조기술구건중조선병독재체pGSadeno-Akt1+COX-2(pGSadeno-A+C),경매절급측서감정후전염인배신세포HEK293포장성위중조rAd5-A+C선병독,병측정병독적적도.체외전염인위암세포주SGC-7901후,Real-time PCR화단백인적분별검측Akt1화COX-2 mRNA화단백질적표체.결과 성공구건rAd5-A+C중조선병독,측정포장적병독적도위1.0×1010pfu/ml.전염조Akt1화COX-2 mRNA표체명현하조,기△Ct치분별시(12.26±0.05)화(5.41±0.09),비rAd5-HK공재조(10.63±0.02)、(3.75±0.08)화대조조(10.57±0.02)、(3.73±0.08)증고(P<0.01),이공재조화대조조비교ΔCt치무명현변화(P>0.05).동양전염조Akt1화COX-2단백표체량여공재조화대조조비교분별하조70.5%화63.7%(P<0.01),공재조화대조조비교Akt1화COX-2단백표체차이무통계학의의(P>0.05).결론 파향성Akt1화COX-2적shRNA선병독재체가이특이성억제Akt1화COX-2적표체,가능성위위암파향성Akt1화COX-2기인치료적신책략.
Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting protein kinase BI (PKB1/Akt1) and cyclooxygenase-2 (COX-2) and observe their expression in human gastric carcinoma cell line SGC-7901. Methods Akt1 and COX-2 shRNA expression frames were sub-cloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno-Aktl + COX-2 ( pGSadeno-A + C) vector. Furthermore after screening and amplification,recombinant ade-novirus vector was digested with Pacl and transfected into HEK293 cells. The replication adenovirus rAd5-A + C was packed and amplified in the HEK293 cells, and its titer was detected. After human SGC-7901 cells in vitro were transfected by rAd5-A + C,Akt1 and COX-2 mRNA and protein expression levels were detected by real-time PCR and Western blot respectively. Compared with rAdS-A + C,SGC-7901 and gen-eral rAd5-HK were selected as the negative controls. Results The recombinant adenovirus rAd5-A + C was constructed successfully and its titer reached 1.0 ×1010 pfu/ml. Aktl and COX-2 mRNA expression was downregulated significantly, and their ACt values ( 12.26±0.05 and 5.41±0.09 respectively ) were higher than rAd5-HK group (10.63±0.02 and 3.75 +0.08 respectively) and control group (10.57± 0.02 and 3.73±0.08 respectively) (P <0.01 ). There was no significant difference between rAd5-HK and control groups (P >0.05). Aktl and COX-2 protein expression was downregulated by 70.5% and 63.7% respectively ( P < 0.01 ) in rAd5-HK group as compared with control group ( P > 0.05 ). Conclu-sion The shRNA aclenovirus vector targeting Akt1 and COX-2 can specifically inhibit Akt1 and COX-2 expression,and this may be a new strategy in gastric carcinoma gene therapy targeting Akt1 and COX-2.
