P53和端粒酶反转录酶基因诱导细胞毒性T淋巴细胞对肝癌细胞杀伤活力的研究

摘要

目的：观察P53和端粒酶反转录酶（ TERT）基因特异性细胞毒性T淋巴细胞（ CTL）对肝癌细胞的杀伤活力。方法采用携带P53和TERT基因的腺病毒感染树突状细胞（DC），并将其作为抗原提呈细胞诱导P53和TERT基因特异性CTL，采用流式细胞术检测P53和TERT基因特异性CTL INF-γ和TNF-α分泌情况；采用乳酸脱氢酶法检测P53和TERT基因特异性CTL对肝癌细胞杀伤情况。结果单纯DC细胞诱导CTL、P53基因特异性CTL 及TERT基因特异性CTL中，分泌INF-γ的细胞百分比分别为（5．8±1．6）％、（14．8±3．7）％及（17．4±5．4）％，分泌 TNF-α的细胞百分比分别为（6．1±1．3）％、（10．9±2．5）％及（15．4±3．2）％，P53和TERT基因特异性CTL的 INF-γ和TNF-α分泌水平均较单纯DC细胞诱导CTL高（ P＜0．001）。 P53基因特异性CTL在10∶1、20∶1、40∶1和80∶1效靶比情况下对肝癌细胞的杀伤效率分别为（11．7±3．4）％、（18．4±5．6）％、（28．6±5．4）％和（44．8±7．2）％， TERT基因特异性 CTL在10∶1、20∶1、40∶1和80∶1效靶比情况下对肝癌细胞的杀伤效率分别为（14．5±3．9）％、（21．7±4．5）％、（32．6±6．1）％和（50．8±6．9）％，均较单纯DC细胞诱导产生的CTL对肝癌细胞的杀伤效率[（5．4±1．5）％、（10．8±4．2）％、（17．7±3．9）％、（25．4±5．8）％]显著升高（P＜0．001）。结论 P53和TERT基因诱导产生的CTL具有更强的肝癌细胞杀伤活性，可望成为肝癌有效的过继治疗手段。%Objective To observe the cancer killing activity of specific cytotoxic T lymphocytes induced by P 53 and telomerase reverse transcriptase(TERT) genes for hepatic carcinoma cells .Methods Dendritic cells(DC) were infected by adenovirus carrying P53 and TERT genes.The adenovirus-infected DCs were used as antigen-presenting cells and induced P 53 and TERT gene-specific cytotoxic T lymphocyte(CTL).The proportions of P53 and TERT gene-specific CTL secreting interferon (IFN)-γand tumor necrosis factor (TNF)-αwere detected by cytometry .The cancer killing activity of P 53 and TERT gene-specific CTL was detected using lactate dehydrogenase assay . Results For CTL induced by DC alone,specific CTL induced by P53 gene and specific CTL induced by TERT gene ,the proportions of cells secreting IFN-γwere (5.8 ±1.6)%,(14.8 ±3.7)%and (17.4 ±5.4)%,respectively,and the proportions of cells secreting TNF-αwere (6.1 ±1.3)%,(10.9 ±2.5)%and (15.4 ±3.2)%,respectively.The levels of cells secreting IFN-γand TNF-αin P53 and TERT gene-specific CTL were higher than those in CTL induced by DC alone(P<0.001).The cancer cell killing efficiencies of P53 gene-specific CTL with the ratio of 10∶1,20∶1,40∶1 and 80∶1 were (11.7 ±3.4)%,(18.4 ±5.6)%,(28.6 ±5.4)%and (44.8 ±7.2)%,respectively,and the cancer cell killing efficiencies of TERT gene-specific CTL with the ratio of 10 ∶1,20 ∶1,40 ∶1 and 80 ∶1 were (14.5 ±3.9)%,(21.7 ±4.5)%, (32.6 ±6.1)% and (50.8 ±6.9)%,respectively,which were significantly higher compared to those of CTL induced by DC alone ((5.4 ±1.5)%,(10.8 ±4.2)%,(17.7 ±3.9)%and (25.4 ±5.8)%,respectively,P<0.001).Conclusion CTLs induced by P53 and TERT genes have more stronger killing activity for hepatic carcinoma cells ,and it is expected to be an effective approach for hepatic cancer.