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目的检测转基因马铃薯体系中表达的人白介素-12(human interleukin 12,hIL-12)的肿瘤杀伤活性。方法通过ELISA法检测转基因马铃薯中hIL-12刺激外周血单个核细胞(PBMC)产生IFN-γ的量以反映生物学活性,并观察hIL-12单克隆抗体对此生物活性的阻断效应;MTT法检测经转基因马铃薯中hIL-12作用的PBMC对NK敏感肿瘤细胞K562的杀伤作用,肿瘤细胞凋亡以Annexin V/PI双染后激光共聚焦显微镜观察。结果转基因马铃薯叶和块茎中表达的hIL-12与重组人白介素-12(rhIL-12)标准品在25～400 pg/ml间均能诱生IFN-γ,其中转基因马铃薯叶和块茎中hIL-12刺激PBMC产生IFN-γ的量分别为(163.34±22.17)～(12 107.10±3 996.51)pg/ml和(224.20±25.76)～(11 196.30±4 067.48)pg/ml,与野生对照组均有统计学差异(P<0.01),而hIL-12单克隆抗体能完全阻断此诱生IFN-γ效应。同时200 pg/ml转基因马铃薯叶、块茎中提取的hIL-12对人红白血病细胞K562的杀伤率分别为(28.4±8.7)%、(32.3±6.9)%,而与野生对照组(10.3±4.9)%的杀伤率相比,有统计学差异(P<0.05)。激光共聚焦显微镜观察到经转基因马铃薯表达hIL-12作用的K562细胞出现早、中、晚各期的凋亡特征。结论转基因马铃薯表达的hIL-12具有诱导PBMC产生IFN-γ及杀伤肿瘤细胞K562的生物学活性。
Objective To detect the tumor-killing activity of human interleukin 12 (hIL-12) expressed in transgenic potato system. Methods The amount of IFN-γ produced by hIL-12-stimulated peripheral blood mononuclear cells (PBMC) in transgenic potato was measured by ELISA to reflect the biological activity and to observe the blocking effect of hIL-12 monoclonal antibody on this biological activity. MTT Method to detect the killing effect of hIL-12-treated PBMC on NK-sensitive tumor cell K562 in transgenic potato. The apoptosis of tumor cells was observed by laser confocal microscopy with Annexin V / PI double staining. Results IFN-γ was induced in hPG-hIL-12 and rhIL-12 from 25 to 400 pg / ml in transgenic potato leaves and tubers, and hIL- 12 stimulated the production of IFN-γ by PBMC from 163.34 ± 22.17 to 12 107.10 ± 3 996.51 pg / ml and from 224.20 ± 25.76 to 11 196.30 ± 4 067.48 pg / ml, respectively, compared with the wild control group There was a significant difference (P <0.01), while hIL-12 monoclonal antibody completely blocked this induced IFN-γ effect. At the same time, hIL-12 extracted from leaves and tubers of 200 pg / ml transgenic potato were (28.4 ± 8.7)% and (32.3 ± 6.9)%, respectively, compared with wild-type control )% Killing rate compared to a statistically significant difference (P <0.05). Laser scanning confocal microscopy showed that K562 cells expressed hIL-12 in transgenic potato appeared early, middle and late stages of apoptosis. Conclusion The transgenic potato expressed hIL-12 has the biological activity of inducing PBMC to produce IFN-γ and kill the tumor cell K562.