重组免疫毒素hIL-2-LuffinP1对淋巴细胞体外增殖的影响

目的检测基因工程免疫毒素hIL-2-LuffinP1的体外生物学活性。方法采用分离的C57和BALB/c小鼠脾细胞进行混合淋巴细胞反应,刀豆球蛋白A(ConA)刺激BALB/c小鼠脾细胞进行淋巴细胞增殖实验,以LuffinP1毒素分子作为对照。3H-TdR核素掺入法检测hIL-2-LuffinP1对淋巴细胞增殖的抑制作用。以CTLL-2细胞检测hIL-2-LuffinP1对IL-2依赖细胞株的杀伤作用,实验共设6组:PBS、IL-2、LuffinP1、LuffinP1 IL-2、hIL-2-LuffinP1、hIL-2-LuffinP1 IL-2,CTLL-2细胞加入各组试剂培养12h,台盼蓝染色,计数每100个细胞中的死亡细胞数。结果随剂量增加,hIL-2-LuffinP1对淋巴细胞增殖的抑制作用逐渐增强,在10-6mol/L浓度时,抑制率可达到97%,而LuffinP1对淋巴细胞增殖无明显抑制作用。hIL-2-LuffinP1对Con A刺激的脾细胞活化也有抑制作用,与在混合淋巴细胞体系中一致,而LuffinP1对上述两个反应体系中的淋巴细胞增殖均无明显抑制作用。加入hIL-2-LuffinP1 IL-2和hIL-2-LuffinP1后CTLL-2细胞的死亡率分别为29.6%和47.4%,明显高于IL-2组(5.6%,P<0.01)。结论重组免疫毒素hIL-2-LuffinP1在体外能够抑制淋巴细胞增殖,对表达IL-2R的活化淋巴细胞产生定向杀伤作用。

Effects of recombinant immunotoxin hIL-2-LuffinP1 on lymphocyte proliferation in vitro

Objective To observe the biological activity of the recombinant immunotoxin hIL-2-LuffinP1 derived by gene engineering in vitro.Methods To observe the inhibitory effect on lymphocytes in vitro,the splenocytes isolated from C57 and BALB/c mice were used for mixed mouse lymphocyte response(MLR),and the lymphocyte proliferation was observed in vitro with BALB/c mice splenocytes stimulated by concanavalin A(ConA).Different concentrations of hIL-2-LuffinP1 was added into the reaction system,with LuffinP1 adopted as control.3H-TdR incorporation assay was used to detect the effects of hIL-2-LuffinP1 on lymphocyte proliferation,and its effects on CTLL-2 cells(IL-2 dependent cells) were also observed.The experiments consisted of six groups(PBS,IL-2,LuffinP1,LuffinP1 IL-2,hIL-2-LuffinP1 and hIL-2-LuffinP1 IL-2).CTLL-2 cells were cultured for 12h after being treated with different agents,and then the cells were stained by trypan blue to estimate the dead cells in every 100 cells.Results It was showed that CPM declined correspondingly to the gradually increased concentration of hIL-2-LuffinP1,and cell proliferation was inhibited obviously.The inhibition rate was up to 97% at the concentration of 10-6mmol/L of hIL-2-LuffinP1.The activity of inhibiting lymphocyte proliferation was proportional to the dosage.On the contrary,no obvious inhibition to lymphocyte proliferation was found in the control group treated with LuffinP1,the inhibition ratio only reached to 14% with the same concentration of hIL-2-LuffinP1.It was also found that,when hIL-2-LuffinP1 and LuffinP1 in different concentrations added to spleen cells stimulated by ConA,hIL-2-LuffinP1 still exhibited strong ability of inhibition on the splenocyte proliferation,while LuffinP1 has no obvious inhibiting effect on the lymphocyte proliferation in the two reaction systems mentioned above.MLR showed the same results.Furthermore,in the experiment of cytotoxic effects of IL-2-LuffinP1 on CTLL-2 cells,the inhibition rates of hIL-2-LuffinP1 IL-2 group and hIL-2-LuffinP1 group were 29.6% and 47.4% respectively,the difference was significant compared with IL-2 group(P<0.01),implying that IL-2-LuffinP1 could kill CTLL-2 cells and its effects could be antagonized by IL-2.Conclusion The results indicate that the recombinant immunotoxin hIL-2-LuffinP1 can inhibit lymphocyte proliferation obviously.It can specifically kill activated T lymphocytes which express IL-2R.