中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
Chinese Journal of Experimental Surgery
2015年
10期
2360-2364
,共5页
高洋%朱亚运%黄新余%郑起%袁周
高洋%硃亞運%黃新餘%鄭起%袁週
고양%주아운%황신여%정기%원주
癌,肝细胞%黏着斑激酶%短发夹RNA%基因治疗%溶瘤腺病毒
癌,肝細胞%黏著斑激酶%短髮夾RNA%基因治療%溶瘤腺病毒
암,간세포%점착반격매%단발협RNA%기인치료%용류선병독
Carcinoma,hepatocellular%Focal adhesion kinase%Short hairpin RNA%Gene therapy%Oncolytic adenovirus
目的 观察人端粒酶反转录酶(hTERT)与杂合启动子HREAF双调控并携带靶向干扰黏着斑激酶(FAK)短发夹RNA(FAK shRNA)的溶瘤腺病毒SG505-siFAK在体外和体内对肝癌的抗癌作用.方法 利用半数细胞培养物感染量(TCID50)法检测重组溶瘤腺病毒SG505、SG505-增强型绿色荧光蛋白(EGFP)和SG505-siFAK病毒在不同细胞中的扩增倍数.利用Western blot检测FAK、早期蛋白E1A和早期蛋白E1B的表达强度.建立裸鼠移植瘤模型,给予溶瘤腺病毒治疗,观察比较肿瘤生长体积,分析FAK免疫组织化学结果.结果 TCID50法检测病毒扩增结果显示:SG505-siFAK在Hep3B和HepG2细胞中的扩增倍数为1.36×108倍和2.02×108倍,明显强于在SMMC-7721、BJ、L02、PANC-1和H460细胞中的扩增能力.SG505-siFAK携带的靶基因FAK-shRNA可以有效降低FAK的表达.Westem blot结果显示SG505、SG505-EGFP和SG505-siFAK感染BJ、L02后E1A、E1B表达较弱,而感染Hep3B、HepG2后E1A、E1B表达较强.噻唑蓝(MTT)法检测细胞增殖结果显示,SG505-siFAK感染Hep3B、HepG2、SMMC-7721、L02、PANC-1和H460的半数抑制浓度(IC50)分别是(0.092 ±0.009)、(0.424±0.414)、(14.790±2.520)、(48.709±0.927)、(5.970±0.945)和(2.710 ±0.244) pfu/cell.裸鼠移植瘤实验结果显示:静脉注射和瘤内注射Wad5、SG505和SG505-siFAK的产生的抑瘤率分别是80.06％、54.14％、79.63％和84.54％、56.64％、87.14％,瘤内注射SG505-siFAK较静脉注射产生更高的抑瘤率(P＜0.01).免疫组织化学结果显示SG505-siFAK可以有效降低FAK在肿瘤组织的表达.结论 SG505-siFAK能选择性地在甲胎蛋白(AFP)阳性肝癌细胞内扩增并产生溶瘤作用,腺病毒SG505自身的溶瘤作用和FAK-shRNA的抑瘤作用表现出协同抗肿瘤效应.
目的 觀察人耑粒酶反轉錄酶(hTERT)與雜閤啟動子HREAF雙調控併攜帶靶嚮榦擾黏著斑激酶(FAK)短髮夾RNA(FAK shRNA)的溶瘤腺病毒SG505-siFAK在體外和體內對肝癌的抗癌作用.方法 利用半數細胞培養物感染量(TCID50)法檢測重組溶瘤腺病毒SG505、SG505-增彊型綠色熒光蛋白(EGFP)和SG505-siFAK病毒在不同細胞中的擴增倍數.利用Western blot檢測FAK、早期蛋白E1A和早期蛋白E1B的錶達彊度.建立裸鼠移植瘤模型,給予溶瘤腺病毒治療,觀察比較腫瘤生長體積,分析FAK免疫組織化學結果.結果 TCID50法檢測病毒擴增結果顯示:SG505-siFAK在Hep3B和HepG2細胞中的擴增倍數為1.36×108倍和2.02×108倍,明顯彊于在SMMC-7721、BJ、L02、PANC-1和H460細胞中的擴增能力.SG505-siFAK攜帶的靶基因FAK-shRNA可以有效降低FAK的錶達.Westem blot結果顯示SG505、SG505-EGFP和SG505-siFAK感染BJ、L02後E1A、E1B錶達較弱,而感染Hep3B、HepG2後E1A、E1B錶達較彊.噻唑藍(MTT)法檢測細胞增殖結果顯示,SG505-siFAK感染Hep3B、HepG2、SMMC-7721、L02、PANC-1和H460的半數抑製濃度(IC50)分彆是(0.092 ±0.009)、(0.424±0.414)、(14.790±2.520)、(48.709±0.927)、(5.970±0.945)和(2.710 ±0.244) pfu/cell.裸鼠移植瘤實驗結果顯示:靜脈註射和瘤內註射Wad5、SG505和SG505-siFAK的產生的抑瘤率分彆是80.06％、54.14％、79.63％和84.54％、56.64％、87.14％,瘤內註射SG505-siFAK較靜脈註射產生更高的抑瘤率(P＜0.01).免疫組織化學結果顯示SG505-siFAK可以有效降低FAK在腫瘤組織的錶達.結論 SG505-siFAK能選擇性地在甲胎蛋白(AFP)暘性肝癌細胞內擴增併產生溶瘤作用,腺病毒SG505自身的溶瘤作用和FAK-shRNA的抑瘤作用錶現齣協同抗腫瘤效應.
목적 관찰인단립매반전록매(hTERT)여잡합계동자HREAF쌍조공병휴대파향간우점착반격매(FAK)단발협RNA(FAK shRNA)적용류선병독SG505-siFAK재체외화체내대간암적항암작용.방법 이용반수세포배양물감염량(TCID50)법검측중조용류선병독SG505、SG505-증강형록색형광단백(EGFP)화SG505-siFAK병독재불동세포중적확증배수.이용Western blot검측FAK、조기단백E1A화조기단백E1B적표체강도.건립라서이식류모형,급여용류선병독치료,관찰비교종류생장체적,분석FAK면역조직화학결과.결과 TCID50법검측병독확증결과현시:SG505-siFAK재Hep3B화HepG2세포중적확증배수위1.36×108배화2.02×108배,명현강우재SMMC-7721、BJ、L02、PANC-1화H460세포중적확증능력.SG505-siFAK휴대적파기인FAK-shRNA가이유효강저FAK적표체.Westem blot결과현시SG505、SG505-EGFP화SG505-siFAK감염BJ、L02후E1A、E1B표체교약,이감염Hep3B、HepG2후E1A、E1B표체교강.새서람(MTT)법검측세포증식결과현시,SG505-siFAK감염Hep3B、HepG2、SMMC-7721、L02、PANC-1화H460적반수억제농도(IC50)분별시(0.092 ±0.009)、(0.424±0.414)、(14.790±2.520)、(48.709±0.927)、(5.970±0.945)화(2.710 ±0.244) pfu/cell.라서이식류실험결과현시:정맥주사화류내주사Wad5、SG505화SG505-siFAK적산생적억류솔분별시80.06％、54.14％、79.63％화84.54％、56.64％、87.14％,류내주사SG505-siFAK교정맥주사산생경고적억류솔(P＜0.01).면역조직화학결과현시SG505-siFAK가이유효강저FAK재종류조직적표체.결론 SG505-siFAK능선택성지재갑태단백(AFP)양성간암세포내확증병산생용류작용,선병독SG505자신적용류작용화FAK-shRNA적억류작용표현출협동항종류효응.
Objective To study the antitumoeffecton the livecancecellof the novel oncolytiadenoviruSG505-short-hairpin RNtargeting the focal adhesion kinase (shFAK), recombinanadenoviruwith the E1gene driven by human telomerase transcriptase (hTERT) promoteand E1gene driven by hybrid HREAF promoteand carrying shFAK.MethodThe amplificationof the virusewere measured by the 50％ tissue culture infective dose (TCID50) assay and the cell viabilitiewere analyzed by methyl thiazol tetrazolium (MTT) assay.The expression of FAK, E1and E1were analyzed by the Western blotting.The nude mice bearing hepatocellulacarcinomwere established and treated by variouoncolytiadenoviruses.The volumeof the tumorwere observed once every week and the FAK expressionin the tumotissue were examined by immunohistochemistry.ResultTCID50 assay revealed the replicationof SG505-siFAK in Hep3and HepG2 were 1.36 × 108 and 2.02 × 108 fold which were more potenthan those in the SMMC-7721, BJ, L02, PANC-1, and H460.The targeted gene FAK-shRNarmed in the SG505-siFAK could down-regulate FAK expression in the Hep3and HepG2.E1and E1expression determined by the Western blotting were weak in the BJ and L02 infected by the SG505, SG505-EGFP and SG505-siFAK and E1and E1expression were strong in the Hep3and HepG2 infected by the SG505, SG505-EGFP and SG505-siFAK.The 50％ inhibitory dose (IC50) value(resulting in 50％ of cell growth inhibition rate) of SG505-siFAK were (0.092 ± 0.009), (0.424 ± 0.414),(14.790-± 2.520), (48.709 ± 0.927), (5.970 ± 0.945), and (2.710 ± 0.244) pfu/cell in the Hep3B, HepG2, SMMC-7721, L02, PANC-1, and H460 cells, respectively.In the xenograftumomodel, the tumoinhibition rateof Wad5, SG505, and SG505-siFAK by intravenouinjection and intratumoral injection were 80.06％, 54.14％, 79.63％ and 84.54％, 56.64％, 87.14％, respectively.Tumortreated with SG505-siFAK by intratumoral injection produced highetumoinhibition ratecomparing to the intravenouinjection (P ＜ 0.01).The immunohistochemistry resultrevealed thathe SG505-siFAK could significantly down-regulate the FAK expression in both groups.Conclusion SG505-siFAK wanovel dual-regulated oncolytiadenoviruand showed selective replication capability and antitumoeffectin the AFP-positive livecancecelland lysed the tumocellwith minimum adverse effectto the normal cell.The oncolytieffectof SG505 and tumoinhibition effectof FAK-shRNwere comprehensive and synergetic.
