中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2011年
4期
245-250
,共6页
孙文洁%熊杰%王伟锋%廖正凯%周福祥%周云峰
孫文潔%熊傑%王偉鋒%廖正凱%週福祥%週雲峰
손문길%웅걸%왕위봉%료정개%주복상%주운봉
人端粒酶逆转录酶%辐射诱导元件%人巨细胞病毒%嵌合启动子%辣根过氧化物酶%吲哚乙酸%肿瘤细胞系
人耑粒酶逆轉錄酶%輻射誘導元件%人巨細胞病毒%嵌閤啟動子%辣根過氧化物酶%吲哚乙痠%腫瘤細胞繫
인단립매역전록매%복사유도원건%인거세포병독%감합계동자%랄근과양화물매%신타을산%종류세포계
Human telomerase reverse transcriptase (hTERT)%Radiation-responsive CArG elements%Cytomegalovirus%Chimeric promoter%Horseradish peroxidase%Prodrug indole-3-acetic acid%Cancer cell lines
目的 探讨放射线联合新型嵌合启动子介导辣根过氧化物酶/吲哚乙酸(HRP/IAA)自杀基因系统特异高效的抗肿瘤作用.方法 构建含有4个串联放射反应元件CArG、含或不含巨细胞病毒(CMV)启动子的人端粒酶逆转录酶嵌合启动子,筛选肿瘤特异性及放射诱导性强的嵌合启动子,下游连接自杀基因辣根过氧化物酶(HRP),检测放射线联合基因治疗对肿瘤细胞HeLa、A549和MHCC97增殖及凋亡的影响.结果 人胚肺成纤维细胞MRC-5中C4-hTC嵌合启动子的活性(0.1±0.0)明显低于肿瘤细胞株HeLa、A549和MHCC97(0.6±0.0、1.1±0.1和1.0±0.1,P＜0.01),经过6 Gy射线诱导后,这种差异更加显著(活性分别为0.2±0.1、1.7±0.2、2.3±0.2和2.3±0.1,P＜0.01).在肿瘤细胞株HeLa、A549和MHCC97中,嵌合启动子C4-hTC-HRP携带的自杀基因系统SER值分别为2.64、2.75和2.82,显著高于单纯hTERT启动子.在肿瘤细胞中,除阴性对照质粒pGL3-control-Luc外,自杀基因与放射治疗相联合对肿瘤细胞的生长抑制明显高于单一治疗方法,而联合组中pC4-hTC-HRP/IAA系统与放射联合的生长抑制作用最显著,对HeLa、A549和MHCC97细胞的生长抑制率分别为67.3%、69.0%和64.6%.在肿瘤细胞中,除阴性对照质粒pGL3-control-Luc外,联合组诱导的早期凋亡率明显高于单一治疗方法组,其中pC4-hTC-HRP/IAA系统与放射联合诱导的凋亡率最高,HeLa、A549和MHCC97细胞凋亡率分别为39.6%、33.0%和33.2%.结论 新型嵌合启动子C4-hTC具有良好的肿瘤特异性及放射诱导性,放射线联合基因治疗对肿瘤细胞具有特异高效的杀伤作用,在肿瘤的基因放疗中具有较强的应用潜力.
目的 探討放射線聯閤新型嵌閤啟動子介導辣根過氧化物酶/吲哚乙痠(HRP/IAA)自殺基因繫統特異高效的抗腫瘤作用.方法 構建含有4箇串聯放射反應元件CArG、含或不含巨細胞病毒(CMV)啟動子的人耑粒酶逆轉錄酶嵌閤啟動子,篩選腫瘤特異性及放射誘導性彊的嵌閤啟動子,下遊連接自殺基因辣根過氧化物酶(HRP),檢測放射線聯閤基因治療對腫瘤細胞HeLa、A549和MHCC97增殖及凋亡的影響.結果 人胚肺成纖維細胞MRC-5中C4-hTC嵌閤啟動子的活性(0.1±0.0)明顯低于腫瘤細胞株HeLa、A549和MHCC97(0.6±0.0、1.1±0.1和1.0±0.1,P＜0.01),經過6 Gy射線誘導後,這種差異更加顯著(活性分彆為0.2±0.1、1.7±0.2、2.3±0.2和2.3±0.1,P＜0.01).在腫瘤細胞株HeLa、A549和MHCC97中,嵌閤啟動子C4-hTC-HRP攜帶的自殺基因繫統SER值分彆為2.64、2.75和2.82,顯著高于單純hTERT啟動子.在腫瘤細胞中,除陰性對照質粒pGL3-control-Luc外,自殺基因與放射治療相聯閤對腫瘤細胞的生長抑製明顯高于單一治療方法,而聯閤組中pC4-hTC-HRP/IAA繫統與放射聯閤的生長抑製作用最顯著,對HeLa、A549和MHCC97細胞的生長抑製率分彆為67.3%、69.0%和64.6%.在腫瘤細胞中,除陰性對照質粒pGL3-control-Luc外,聯閤組誘導的早期凋亡率明顯高于單一治療方法組,其中pC4-hTC-HRP/IAA繫統與放射聯閤誘導的凋亡率最高,HeLa、A549和MHCC97細胞凋亡率分彆為39.6%、33.0%和33.2%.結論 新型嵌閤啟動子C4-hTC具有良好的腫瘤特異性及放射誘導性,放射線聯閤基因治療對腫瘤細胞具有特異高效的殺傷作用,在腫瘤的基因放療中具有較彊的應用潛力.
목적 탐토방사선연합신형감합계동자개도랄근과양화물매/신타을산(HRP/IAA)자살기인계통특이고효적항종류작용.방법 구건함유4개천련방사반응원건CArG、함혹불함거세포병독(CMV)계동자적인단립매역전록매감합계동자,사선종류특이성급방사유도성강적감합계동자,하유련접자살기인랄근과양화물매(HRP),검측방사선연합기인치료대종류세포HeLa、A549화MHCC97증식급조망적영향.결과 인배폐성섬유세포MRC-5중C4-hTC감합계동자적활성(0.1±0.0)명현저우종류세포주HeLa、A549화MHCC97(0.6±0.0、1.1±0.1화1.0±0.1,P＜0.01),경과6 Gy사선유도후,저충차이경가현저(활성분별위0.2±0.1、1.7±0.2、2.3±0.2화2.3±0.1,P＜0.01).재종류세포주HeLa、A549화MHCC97중,감합계동자C4-hTC-HRP휴대적자살기인계통SER치분별위2.64、2.75화2.82,현저고우단순hTERT계동자.재종류세포중,제음성대조질립pGL3-control-Luc외,자살기인여방사치료상연합대종류세포적생장억제명현고우단일치료방법,이연합조중pC4-hTC-HRP/IAA계통여방사연합적생장억제작용최현저,대HeLa、A549화MHCC97세포적생장억제솔분별위67.3%、69.0%화64.6%.재종류세포중,제음성대조질립pGL3-control-Luc외,연합조유도적조기조망솔명현고우단일치료방법조,기중pC4-hTC-HRP/IAA계통여방사연합유도적조망솔최고,HeLa、A549화MHCC97세포조망솔분별위39.6%、33.0%화33.2%.결론 신형감합계동자C4-hTC구유량호적종류특이성급방사유도성,방사선연합기인치료대종류세포구유특이고효적살상작용,재종류적기인방료중구유교강적응용잠력.
Objective To explore the synergistic anti-tumor effect of radiotherapy and horseradish peroxidase/prodrug indole-3-acetic acid (HRP/IAA) gene therapy system using chimeric hTERT promoter responsive to ionizing radiation.Methods The synthetic hTERT promoters containing four tandem-repeat copies of radio-inducible CArG elements, and the chimeric promoter containing cytomegalovirus (CMV)early promoter were both constructed.The activities of the chimeric promoters in cancer cell lines ( HeLa,A549, and MHCC97 ) and normal cell line ( MRC-5 ) were detected using luciferase reporter gene expression analysis after a 60 Co γ-irradiation treatment at a series of doses (a single dose of 0 to 10 Gy).The anti-tumor effect of combining irradiation with HRP/IAA gene-directed enzyme prodrug therapy system controlled by the chimeric promoter was tested by colony formation assay, cell counting and apoptosis analysis.Results The chimeric promoters were ineffective in normal human cells, even after irradiation, but the expression of luciferase gene in tumor cells was significantly higher.The activity of the chimeric promoter in MRC-5 cells was 22.3%, 12.9% and 13.6% of that in HeLa, A549 and MHCC97 cells, respectively.After irradiation,the ratios were 11.7%, 8.7% and 8.8%, respectively.Furthermore, the chimeric promoters could successfully induce the expression of luciferase gene following different doses of radiation, with maximal inducible activity seen after 6 Gy irradiation.The chimeric promoter containing four tandem-repeat copies of radio-inducible CArG elements and CMV early promoter showed the highest activity with 6 Gy irradiation.The relative luciferase activities in HeLa, A549 and MHCC97 cells were 1.7 ± 0.2, 2.3 ± 0.2 and 2.3±0.1, respectively.The chimeric promoter mediated suicide gene therapy system could increase radiosensitivity in different cancer cells.Compared with the control system, it plus irradiation showed stronger cell proliferation inhibition, 67.3% vs.26.1% in HeLa, 69.0% vs.28.3% in A549, 64.6% vs.20.8% in MHCC97 cells, and also higher apoptosis-inducing effect, 39.6% vs.14.2% in HeLa, 33.0% vs.12.4%in A549, and 33.2% vs.14.2% in MHCC97 cells.Conclusions Chimeric promoter containing hTERT promoter, CArG elements and CMV promoter preserve the tumor-specificity in telomerase-positive tumor cells, and irradiation-responsive to low dose of radiation.The suicide gene therapy using this promoter plus radiotherapy show a strong anti-tumor effect in vitro.It is expected to have a good potential for future application in gene radiotherapy.
