中华消化外科杂志
中華消化外科雜誌
중화소화외과잡지
CHINESE JOURNAL OF DIGESTIVE SURGERY
2011年
4期
286-289
,共4页
万世奇%张太平%王天笑%郑莲芳%李方%李汇华
萬世奇%張太平%王天笑%鄭蓮芳%李方%李彙華
만세기%장태평%왕천소%정련방%리방%리회화
胰腺肿瘤%表皮生长因子受体%重组腺相关病毒载体
胰腺腫瘤%錶皮生長因子受體%重組腺相關病毒載體
이선종류%표피생장인자수체%중조선상관병독재체
Pancreatic neoplasms%Epidermal growth factor receptor%Recombinant adeno-associated virus vector
目的 构建能在体内长期表达抗表皮生长因子受体(EGFR)抗体的重组腺相关病毒(rAAV)载体,观察其对人胰腺癌细胞株的抑制作用.方法 将抗人EGFR单链抗体可变区(SCFV)基因插入腺相关病毒载体的Kpn Ⅰ和BglⅡ位点,构建成含抗EGFR单链抗体基因的重组腺相关病毒载体(rAAV-anfiEGFR).设立对照组(rAAV1-EGFP组)和实验组(rAAV1-anti EGFR组),观察抗EGFR单链抗体蛋白的表达情况.采用Western blot检测抗体蛋白的表达、CCK-8法和流式细胞仪检测6种人胰腺癌细胞株(PCT-3、SW1990、Capan-1、ASPC-1、MiaPaCa-2及PANC-1细胞)的抑制率和凋亡率.两组比较采用t检验.结果 抗EGFR单链抗体蛋白能在6种人胰腺癌细胞株中表达,对照组和实验组胰腺癌ASPC-1细胞抑制率分别为1.1%±2.4%和15.1%±3.5%,两组比较,差异有统计学意义(t=6.598,P＜0.05).对照组和实验组PANC-1细胞凋亡率分别为7.0%±3.0%和11.4%±2.5%,两组比较,差异无统计学意义(t=1.952,P＞0.05);对照组和实验组SW1990、ASPC-1、Capan-1、PCT-3、MiaPaCa-2细胞凋亡率分别为1.1%±0.8%、1.5%±0.7%、1.7%±1.2%、1.1%±0.7%、2.2%±1.1%和17.6%±2.2%、46.9%±3.9%、20.0%±2.8%、12.1%±1.6%、31.1%±2.5%,两组比较,差异均有统计学意义(t=12.208,19.846,10.405,10.909,18.327,P＜0.05).结论 成功构建了携带抗EGFR单链抗体基因的rAAV载体,其表达的抗EGFR单链抗体蛋白对人胰腺癌细胞株有明显的抑制作用.
目的 構建能在體內長期錶達抗錶皮生長因子受體(EGFR)抗體的重組腺相關病毒(rAAV)載體,觀察其對人胰腺癌細胞株的抑製作用.方法 將抗人EGFR單鏈抗體可變區(SCFV)基因插入腺相關病毒載體的Kpn Ⅰ和BglⅡ位點,構建成含抗EGFR單鏈抗體基因的重組腺相關病毒載體(rAAV-anfiEGFR).設立對照組(rAAV1-EGFP組)和實驗組(rAAV1-anti EGFR組),觀察抗EGFR單鏈抗體蛋白的錶達情況.採用Western blot檢測抗體蛋白的錶達、CCK-8法和流式細胞儀檢測6種人胰腺癌細胞株(PCT-3、SW1990、Capan-1、ASPC-1、MiaPaCa-2及PANC-1細胞)的抑製率和凋亡率.兩組比較採用t檢驗.結果 抗EGFR單鏈抗體蛋白能在6種人胰腺癌細胞株中錶達,對照組和實驗組胰腺癌ASPC-1細胞抑製率分彆為1.1%±2.4%和15.1%±3.5%,兩組比較,差異有統計學意義(t=6.598,P＜0.05).對照組和實驗組PANC-1細胞凋亡率分彆為7.0%±3.0%和11.4%±2.5%,兩組比較,差異無統計學意義(t=1.952,P＞0.05);對照組和實驗組SW1990、ASPC-1、Capan-1、PCT-3、MiaPaCa-2細胞凋亡率分彆為1.1%±0.8%、1.5%±0.7%、1.7%±1.2%、1.1%±0.7%、2.2%±1.1%和17.6%±2.2%、46.9%±3.9%、20.0%±2.8%、12.1%±1.6%、31.1%±2.5%,兩組比較,差異均有統計學意義(t=12.208,19.846,10.405,10.909,18.327,P＜0.05).結論 成功構建瞭攜帶抗EGFR單鏈抗體基因的rAAV載體,其錶達的抗EGFR單鏈抗體蛋白對人胰腺癌細胞株有明顯的抑製作用.
목적 구건능재체내장기표체항표피생장인자수체(EGFR)항체적중조선상관병독(rAAV)재체,관찰기대인이선암세포주적억제작용.방법 장항인EGFR단련항체가변구(SCFV)기인삽입선상관병독재체적Kpn Ⅰ화BglⅡ위점,구건성함항EGFR단련항체기인적중조선상관병독재체(rAAV-anfiEGFR).설립대조조(rAAV1-EGFP조)화실험조(rAAV1-anti EGFR조),관찰항EGFR단련항체단백적표체정황.채용Western blot검측항체단백적표체、CCK-8법화류식세포의검측6충인이선암세포주(PCT-3、SW1990、Capan-1、ASPC-1、MiaPaCa-2급PANC-1세포)적억제솔화조망솔.량조비교채용t검험.결과 항EGFR단련항체단백능재6충인이선암세포주중표체,대조조화실험조이선암ASPC-1세포억제솔분별위1.1%±2.4%화15.1%±3.5%,량조비교,차이유통계학의의(t=6.598,P＜0.05).대조조화실험조PANC-1세포조망솔분별위7.0%±3.0%화11.4%±2.5%,량조비교,차이무통계학의의(t=1.952,P＞0.05);대조조화실험조SW1990、ASPC-1、Capan-1、PCT-3、MiaPaCa-2세포조망솔분별위1.1%±0.8%、1.5%±0.7%、1.7%±1.2%、1.1%±0.7%、2.2%±1.1%화17.6%±2.2%、46.9%±3.9%、20.0%±2.8%、12.1%±1.6%、31.1%±2.5%,량조비교,차이균유통계학의의(t=12.208,19.846,10.405,10.909,18.327,P＜0.05).결론 성공구건료휴대항EGFR단련항체기인적rAAV재체,기표체적항EGFR단련항체단백대인이선암세포주유명현적억제작용.
Objective To construct a recombinant adeno-associated virus(rAAV)vector containing a human anti-epidermal growth factor receptor(anti-EGFR)single-chain variable fragment antibody gene,and observe its inhibitory effects on pancreatic cancer cell lines.Methods Human anti-EGFR single-chain variable fragment antibody gene was inserted into the Kpn I and Bgl Ⅱ sites to construct a rAAV-anti EGFR vector,and then rAAV1-EGFP group and rAAV1-anti EGFR group were established.The expression of anti-EGFR antibody was observed.Antibody expression was detected by Western blot,and the inhibition and apoptosis rates of human pancreatic cancer cell lines(PCT-3,SW1990,Capan-1,ASPC-1,MiaPaCa-2 and PANC-1 cells)were detected by CCK-8 assay and flow cytometry,respectively.All data were analyzed using the t test.Results The results of Western blot assay demonstrated that anti-EGFR antibody was expressed in 6 pancreatic cancer cell lines.The inhibition rates of rAAV1-EGFP and rAAVl-anti EGFR on pancreatic ASPC-1 cells were 1.1%± 2.4% and 15.1%±3.5%,respectively,with a significant difference between the 2 groups(t =6.598,P ＜0.05).The apoptosis rates of PANC-1 cells were 7.0% ± 3.0% in the rAAV1-EGFP group and 1 1.4% ± 2.5% in the rAAV1-anti EGFR group,with no significant difference between the 2 grouvs(t = 1.952,P ＞0.05).The apoptosis rates of SW1990,ASPC-1,Capan-1,PCT-3,MiaPaCa-2 cells were 1.1% ± 0.8%,1.5% ± 0.7%,1.7% ± 1.2%,1.1%±0.7% and 2.2% ± 1.1% in the rAAV1-EGFP group,and 17.6% ± 2.2%,46.9% ± 3.9%,20.0% ±2.8%,12.1% ± 1.6% and 31.1% ±2.5% in the rAAV1-anti EGFR group,respectively,with significant differences between the 2 groups(t = 12.208,19.846,10.405,10.909,18.327,P ＜0.05).Conclusions A rAAV-anti EGFR vector with human anti-EGFR single-chain variable fragment antibody gene was constructed.Anti-EGFR antibody has obvious inhibition effects on pancreatic cancer cell lines.
