中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2008年
5期
355-359
,共5页
张剑%甘愉%胡晶莹%赵新泰
張劍%甘愉%鬍晶瑩%趙新泰
장검%감유%호정형%조신태
癌,肝细胞%基因,抑制,肿瘤%腺病毒载体
癌,肝細胞%基因,抑製,腫瘤%腺病毒載體
암,간세포%기인,억제,종류%선병독재체
Carcinoma,hepatocellulal%Genes,suppressor,tumor%Adenovirus vector
目的 建立肝癌抑制基因-1(HCCS1)肿瘤靶向性表达载体,提高肿瘤治疗的安全性.方法 活细胞计数试剂盒测定HCCS1高表达对正常细胞和肿瘤细胞的影响,荧光素酶试验检测肿瘤特异性启动子PEG-3p在正常肝细胞和肝癌细胞中的相对转录活性,AdEasyTM系统包装并利用PCR鉴定重组腺病毒Ad-PEG-3p-HCCS1,Western blot检测重组腺病毒感染后HCCS1在正常细胞和肿瘤细胞中的表达情况,结晶紫试验和四甲基偶氮唑盐试验观察该重组腺病毒体外抗肿瘤的靶向性. 结果 HCCS1高表达对肿瘤细胞株BEL-7404和SW-620的生长抑制作用明显超过正常细胞株L02和正常人肺成纤维细胞,96 h抑制率达60%.荧光素酶试验显示,PEG-3p在BEL-7404、BEL-7405、QGY-7703中的相对转录活性分别为L02的3.9、4.7、1.5倍.成功构建了新抑癌基因HCCS1肿瘤靶向性表达的重组腺病毒Ad-PEG-3p-HCCS1,Western blot检测结果显示,在BEL7404和QGY-7703中,HCCS1表达高于在L02中的表达.结晶紫试验和四甲基偶氮唑盐试验显示,Ad-PEG-3p-HCCS1与Ad-CMV-HCCS1相比,在不降低对肿瘤抑制效果的前提下,明显降低了对正常细胞的杀伤作用. 结论 肿瘤细胞对HCCS1的生长抑制作用更为敏感,PEG-3p在肝癌细胞中也具有肿瘤特异性,Ad-PEG-3p-HCCS1可特异性地在肿瘤细胞中表达HCCS1,从而提高HCCS1基因治疗的安全性.
目的 建立肝癌抑製基因-1(HCCS1)腫瘤靶嚮性錶達載體,提高腫瘤治療的安全性.方法 活細胞計數試劑盒測定HCCS1高錶達對正常細胞和腫瘤細胞的影響,熒光素酶試驗檢測腫瘤特異性啟動子PEG-3p在正常肝細胞和肝癌細胞中的相對轉錄活性,AdEasyTM繫統包裝併利用PCR鑒定重組腺病毒Ad-PEG-3p-HCCS1,Western blot檢測重組腺病毒感染後HCCS1在正常細胞和腫瘤細胞中的錶達情況,結晶紫試驗和四甲基偶氮唑鹽試驗觀察該重組腺病毒體外抗腫瘤的靶嚮性. 結果 HCCS1高錶達對腫瘤細胞株BEL-7404和SW-620的生長抑製作用明顯超過正常細胞株L02和正常人肺成纖維細胞,96 h抑製率達60%.熒光素酶試驗顯示,PEG-3p在BEL-7404、BEL-7405、QGY-7703中的相對轉錄活性分彆為L02的3.9、4.7、1.5倍.成功構建瞭新抑癌基因HCCS1腫瘤靶嚮性錶達的重組腺病毒Ad-PEG-3p-HCCS1,Western blot檢測結果顯示,在BEL7404和QGY-7703中,HCCS1錶達高于在L02中的錶達.結晶紫試驗和四甲基偶氮唑鹽試驗顯示,Ad-PEG-3p-HCCS1與Ad-CMV-HCCS1相比,在不降低對腫瘤抑製效果的前提下,明顯降低瞭對正常細胞的殺傷作用. 結論 腫瘤細胞對HCCS1的生長抑製作用更為敏感,PEG-3p在肝癌細胞中也具有腫瘤特異性,Ad-PEG-3p-HCCS1可特異性地在腫瘤細胞中錶達HCCS1,從而提高HCCS1基因治療的安全性.
목적 건립간암억제기인-1(HCCS1)종류파향성표체재체,제고종류치료적안전성.방법 활세포계수시제합측정HCCS1고표체대정상세포화종류세포적영향,형광소매시험검측종류특이성계동자PEG-3p재정상간세포화간암세포중적상대전록활성,AdEasyTM계통포장병이용PCR감정중조선병독Ad-PEG-3p-HCCS1,Western blot검측중조선병독감염후HCCS1재정상세포화종류세포중적표체정황,결정자시험화사갑기우담서염시험관찰해중조선병독체외항종류적파향성. 결과 HCCS1고표체대종류세포주BEL-7404화SW-620적생장억제작용명현초과정상세포주L02화정상인폐성섬유세포,96 h억제솔체60%.형광소매시험현시,PEG-3p재BEL-7404、BEL-7405、QGY-7703중적상대전록활성분별위L02적3.9、4.7、1.5배.성공구건료신억암기인HCCS1종류파향성표체적중조선병독Ad-PEG-3p-HCCS1,Western blot검측결과현시,재BEL7404화QGY-7703중,HCCS1표체고우재L02중적표체.결정자시험화사갑기우담서염시험현시,Ad-PEG-3p-HCCS1여Ad-CMV-HCCS1상비,재불강저대종류억제효과적전제하,명현강저료대정상세포적살상작용. 결론 종류세포대HCCS1적생장억제작용경위민감,PEG-3p재간암세포중야구유종류특이성,Ad-PEG-3p-HCCS1가특이성지재종류세포중표체HCCS1,종이제고HCCS1기인치료적안전성.
Objective To construct a tumor-targeting recombinant adeflovims vector containing hepatocellular carcinoma suppressor gene HCCS1 to enhance the safety of tumor treatment.Methods CCK-8 assay was used to observe different inhibitory effects on normal and malignant liver cells with high expressions of HCCS1 protein.The relative transcriptional activity of PEG-3p was quantified by luciferase assay.Recombinant adenovims Ad-PEG-3p-HCCS1 was packaged with AdEasyTM system and confirmed by PCR.The tumor-targeted expression of HCCS1 protein in cells irifected wim Ad-PEG-3p-HCCS1 was determined bv western blot.Crystal violet assay and MTT assay were applied to observe the selective anti-tumor effects of the newly constructed virus in vitro.Results A higher inhibitory rate of about 60%was found in BEL-7404 and SW-620 than that in L02 and NHLF 96 h after the high expression Of HCCS1.Luciferase assay showed 3.9-,4.7-,and 1.5-fold transcriptional activity in BEL-7404,BEL-7405 and QGY-7703 respectively,in comparison wim mat in L02.Ad-PEG-3p-HCCS1 was constructed successfully and was verified by PCR.Western blot indicated that high expression of HCCS1 could be induced in BEL-7404 and QGY-7703 but not in L02.Crystal violet assay and MTT aSSay showed that it remarkably reduced t11e toxicity tO L02 but still had enough antitumoral eflfect on Ad-CMV-HCCS1.Conclusions With high expression of HCCS1 the tumor cells we used ale being inhibited more.PEG-3p has the tumor-selective driving function in malignant liver cells.Our recombinant adenovirus Ad-PEG-3p-HCCS1 can tumor-targetingly induce HCCS1 expression in tumor cells,which Can improve the safety of gene therapy with HCCS1.
