[蛋白酶体抑制剂MG132在诱导白血病细胞共刺激分子CD86表达中的作用及其对同种异体混合淋巴细胞反应的影响]

Zhou Yong-Ming, Guo Wei, Xue Ke-Ying, Zhou Hao, Cheng Yan-Xiang, Guo Tian-Nan, Li Hui-Yu, Huang Shi-Ang

Center for Stem Cell Amplification and Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.

Zhonghua Yi Xue Za Zhi. 2007 Mar 13;87(10):710-3.

OBJECTIVE

To investigate the role of proteasome inhibitors MG132 in the inducing the expression of the costimulatory molecules CD80 and CD86 in leukemia cells and its effect on allogeneic mixed lymphocyte reaction.

METHODS

Acute myelocytic leukemia cells of the line HL-60 and chronic myelocytic leukemia cells of the line K562 were cultured. 7-AAD staining and flow cytometry (FC) were used to examine the viability of the cells. MG132, a proteasome inhibitor, of the concentrations of 2 or 3 micromol/L was added into the culture fluid of HL-60 cells for 24 h and 48 h respectively and then annexin V/7-AAD staining and FC were used to detect the apoptosis of the cells. HL-60 and K562 cells treated with 1 micromol/L MG132 for 24 h and 48 h respectively, anti-CD80 and anti-CD86 antibodies were added, then FC was used to detect the expression of CD80 and CD86. The mRNA expression of CD86 in the HL-60 cells treated with 1 micromol/L MG132 was examined by RT-PCR. HL-60 and K562 cells were treated by 1 micromol/L MG132 for 48 h and then underwent irradiation of 75 Gy Co-60 to kill the cells with their antigenicity preserved. Peripheral blood mononuclear cells (PBMNC) of healthy volunteers, as reactive cells, were isolated and inoculated into the Co-60 treated HL-60 and K532 cells of different concentrations, as stimulating cells, for 5 d, CCK-8, a new agent to detect the cell viability, was added for 4 h, and then the A value of absorbance was measured at the wave length of 450 nm of enzyme labeling instrument. Control groups were set up for all tests.

RESULTS

The cell viability rates of the HL-60 cell treated with 1 micromol/L MG132 for 24 h and 48 h were 92.95% and 85.87% respectively. The apoptotic rats of the HL-60 cells treated with MG132 were increased dose- and time-dependently. Before MG132 treatment K562 cells did not express CD86, and the CD86 expression of the HL-60 cells was up-regulated time-dependently (all P < 0.01). The mRNA expression of CD86 in the HL-60 treated with MG132 was up-regulated time-dependently (P < 0.01). CKK8 test showed that the proliferation level of PBMNC gradually increased along with the concentration of HL-60 cells treated with MG132 and reached its peak when the concentration of the HL-60 cells was 1 x 10(5) (P < 0.01). No remarkable proliferation of PBMNC was seen in the K562 groups no matter if the HL-60 cells had been treated with MG132.

CONCLUSION

MG132 induces the expression of costimulatory molecule CD86 in the HL-60 cells, thus improving the proliferation of PBMNC.

目的

探讨蛋白酶体抑制剂MG132对白血病细胞共刺激分子CD80和CD86表达的诱导作用及其对同种异体混合淋巴细胞反应的影响。

方法

培养人急性髓系白血病HL-60细胞株和慢性髓系白血病K562细胞株。采用7-氨基放线菌素D（7-AAD）染色及流式细胞术（FC）检测细胞活力。将浓度为2或3 μmol/L的蛋白酶体抑制剂MG132分别加入HL-60细胞培养液中作用24 h和48 h，然后采用膜联蛋白V/7-AAD染色及FC检测细胞凋亡情况。HL-60和K562细胞分别用1 μmol/L MG132处理24 h和48 h后，加入抗CD80和抗CD86抗体，再用FC检测CD80和CD86的表达。采用逆转录-聚合酶链反应（RT-PCR）检测1 μmol/L MG132处理的HL-60细胞中CD86的mRNA表达。HL-和K562细胞用1 μmol/L MG132处理48 h后，进行75 Gy 60Co照射，使其细胞死亡但保留抗原性。分离健康志愿者外周血单个核细胞（PBMNC）作为反应细胞，接种于不同浓度经60Co处理的HL-60和K532细胞作为刺激细胞，培养5 d，加入新型细胞活力检测试剂CCK-8作用4 h，然后在酶标仪450 nm波长处测定吸光度A值。所有试验均设对照组。

结果

1 μmol/L MG132处理24 h和48 h的HL-60细胞活力率分别为92.95%和85.87%。MG132处理的HL-60细胞凋亡率呈剂量和时间依赖性增加。MG132处理前K562细胞不表达CD86，HL-60细胞CD86表达呈时间依赖性上调（均P<0.01）。MG132处理的HL-60细胞中CD86的mRNA表达呈时间依赖性上调（P<0.01）。CCK8检测显示，PBMNC的增殖水平随MG132处理的HL-60细胞浓度增加而逐渐升高，当HL-60细胞浓度为1×105时达到峰值（P<0.01）。无论HL-60细胞是否经MG132处理，K562组均未见PBMNC明显增殖。