摘要：
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.%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.
