转基因马铃薯中人白介素-12的生物学活性

Bioactivity of human interleukin-12 from transgenic potatoes

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 bioactivity of human iuterleukin-12 （hlL-12） extracted from transgenic potatoes. Methods ELISA was used to assess hIL-12-induced IFN-γ secretion in peripheral blood mononuclear cells （PBMC） and observe the blocking effect of monoclonal antibody to hIL-12 on the bioactivity of hIL-12. Lethal effect of hIL-12-activated PBMC on NK sensitive I（562 cell line was detected by MTT assay. Apoptosis of tumor cells was observed under laser confocal microscope with double Annexin V/PI staining. Results The expression of hlL-12 and recombinant hIL-12 （rhlL-12） extracted from transgenic potato leaves or tubers at the concentration of 25 pg/ml to 400 pg/ml induced IFN-3, secretion in PBMC. The mount of secreted IFN-γ/was （163.34± 22.17 ）pg/ml to （12 107.10 ±3 996.51 ）pg/ml after treated with hIL-12 from potato leaves and was （224.20 ± 25.76 ） pg/ml to （ 11 196.30 ± 4 067.48 ） pg/ml after treated with potato tubers, which was significantly higher than that of commercially available hlL-12 （P 〈0.01 ）. The secreted IFN-γ/was almost fully blocked by monoclonal hIL-12 antibody. The apoptotic rate of K562 cells treated with 200 pg/ml hIL-12 extracted from transgenic potato leaves or tubers was （28.4 ± 8.7）% and （32.3±6.9）%, respectively, which was much higher than that of commercially available hIL-12 （P 〈 0.05 ）. Laser confocal microscopy showed the characteristic apoptosis of K562 cells at early, middle and late phases after treated with the extracted hIL-12. Conclusion hIL-12 extracted from transgenic potato induces PBMC producing IFN-γ and excert antitumor effect on K562 leukemia cells.