中华肝胆外科杂志
中華肝膽外科雜誌
중화간담외과잡지
CHINESE JOURNAL OF HEPATOBILIARY SURGERY
2011年
6期
488-491
,共4页
方益锋%单云峰%高申孟%张启瑜
方益鋒%單雲峰%高申孟%張啟瑜
방익봉%단운봉%고신맹%장계유
腺病毒%端粒酶逆转录酶%缺氧反应元件%内皮抑素
腺病毒%耑粒酶逆轉錄酶%缺氧反應元件%內皮抑素
선병독%단립매역전록매%결양반응원건%내피억소
Adenovirus%Human telomerase reverse transcriptase%Hypoxia response element%Endostatin
目的 构建新型双重调控肿瘤特异性增殖型腺病毒载体治疗系统.方法 通过基因重组技术构建受端粒酶逆转录酶启动子控制腺病毒E1A表达、缺氧反应元件启动子控制E1B表达并携带人内皮抑素基因的质粒pTPHre-hEndo.将质粒pTPHre-hEndo与含有腺病毒右臂的质粒pB-GHE3共转染至293细胞中同源重组得到带抗肿瘤基因的双重调控增殖型腺病毒AdTPHre-hEndo.用TCID50方法测定病毒滴度.通过增殖实验观察重组病毒的选择性增殖能力.利用ELISA法检测人内皮抑素抗癌基因的表达.结果 成功构建了由双重调控的选择增殖型腺病毒AdTPHre-hEndo,病毒滴度为3.25×1010pfu/ml.增殖实验结果证实AdTPHre-hEndo可以选择性地在端粒酶阳性的胰腺癌细胞中增殖.随着感染时间的延长,肿瘤细胞培养上清液中内皮抑素表达量不断增加,第7天达(310.25±1.41)ng/ml,明显高于携带该基因的非增殖型腺病毒载体(112.53±4.41)ng/ml.结论 AdTPHre-hEndo具有高效表达人内皮抑素和在胰腺癌细胞内增殖的能力,为胰腺癌的生物治疗提供了一种新型的基因-病毒治疗系统.
目的 構建新型雙重調控腫瘤特異性增殖型腺病毒載體治療繫統.方法 通過基因重組技術構建受耑粒酶逆轉錄酶啟動子控製腺病毒E1A錶達、缺氧反應元件啟動子控製E1B錶達併攜帶人內皮抑素基因的質粒pTPHre-hEndo.將質粒pTPHre-hEndo與含有腺病毒右臂的質粒pB-GHE3共轉染至293細胞中同源重組得到帶抗腫瘤基因的雙重調控增殖型腺病毒AdTPHre-hEndo.用TCID50方法測定病毒滴度.通過增殖實驗觀察重組病毒的選擇性增殖能力.利用ELISA法檢測人內皮抑素抗癌基因的錶達.結果 成功構建瞭由雙重調控的選擇增殖型腺病毒AdTPHre-hEndo,病毒滴度為3.25×1010pfu/ml.增殖實驗結果證實AdTPHre-hEndo可以選擇性地在耑粒酶暘性的胰腺癌細胞中增殖.隨著感染時間的延長,腫瘤細胞培養上清液中內皮抑素錶達量不斷增加,第7天達(310.25±1.41)ng/ml,明顯高于攜帶該基因的非增殖型腺病毒載體(112.53±4.41)ng/ml.結論 AdTPHre-hEndo具有高效錶達人內皮抑素和在胰腺癌細胞內增殖的能力,為胰腺癌的生物治療提供瞭一種新型的基因-病毒治療繫統.
목적 구건신형쌍중조공종류특이성증식형선병독재체치료계통.방법 통과기인중조기술구건수단립매역전록매계동자공제선병독E1A표체、결양반응원건계동자공제E1B표체병휴대인내피억소기인적질립pTPHre-hEndo.장질립pTPHre-hEndo여함유선병독우비적질립pB-GHE3공전염지293세포중동원중조득도대항종류기인적쌍중조공증식형선병독AdTPHre-hEndo.용TCID50방법측정병독적도.통과증식실험관찰중조병독적선택성증식능력.이용ELISA법검측인내피억소항암기인적표체.결과 성공구건료유쌍중조공적선택증식형선병독AdTPHre-hEndo,병독적도위3.25×1010pfu/ml.증식실험결과증실AdTPHre-hEndo가이선택성지재단립매양성적이선암세포중증식.수착감염시간적연장,종류세포배양상청액중내피억소표체량불단증가,제7천체(310.25±1.41)ng/ml,명현고우휴대해기인적비증식형선병독재체(112.53±4.41)ng/ml.결론 AdTPHre-hEndo구유고효표체인내피억소화재이선암세포내증식적능력,위이선암적생물치료제공료일충신형적기인-병독치료계통.
Objective To develop a double-regulated replicative adenovirus carrying the Human endostatin gene(hEndo). Methods The plasmid pTPHre-hEndo was constructed by gene engineering technique, carrying human endostatin gene, in which El A gene and E1B gene were driven by human telomerase reverse transcriptase (hTERT) promoter and hypoxia response element (HRE) promoter,respectively. The pTPHre-hEndo was co-transfected with pBHGE3 in 293 cells to generate recombinant adenovirus AdTPHre-hEndo. Virus titer was measured by the TCID50 method. Virus replication assay was performed to evaluate the selective replication ability of AdTPHre-hEndo. The transgene expression of endostatin was detected by ELISA assay. Results A novel gene-viral therapeutic system AdTPHre-hEndo was constructed by gene engineering technique and its titer was 3. 25 X 1010 pfu/ml.Proliferative test revealed that AdTPHre-hEndo could proliferate selectively in telomerase positive tumors. Furthermore, in comparison with non-replicative adenovirus Ad-hEndo, the transgene expression of endostatin mediated with AdTPHre-hEndo was significantly increased (P < 0. 01).Conclusion The novel gene-viral therapeutic system AdTPHre-hEndo has the capacity to replicate in pancreatic cancer cells and expresses the endostatin efficiently, and may provide a new strategy for pancreatic cancer gene therapy.
