摘要
本研究探讨蛋白酶体抑制剂MG132诱导HL-60细胞表达共刺激分子CD80、CD86及其对混合淋巴细胞反应的作用。流式细胞仪检测MG132诱导HL-60细胞表达共刺激分子CD80、CD86及细胞活力;逆转录聚合酶链反应(RT-PCR)分析CD80和CD86 mRNA表达情况;MG132诱导HL-60细胞表达共刺激分子CD86后,用75 Gy Co-60照射HL-60细胞,杀死HL-60细胞,保留抗原性作为刺激细胞,用健康人外周血单个核细胞作为反应细胞,用不同浓度HL-60细胞刺激健康人单个核细胞,HL-60细胞对健康人单个核细胞的增殖作用。结果表明:MG132上调HL-60细胞表达CD86,MG132诱导HL-60细胞的凋亡率呈浓度依耐性和时间依耐性。结论:高浓度的MG132对HL-60细胞有直接杀灭作用,低浓度MG132诱导HL-60细胞表达共刺激分子CD86,对健康人单个核细胞有增殖作用。MG132诱导HL-60细胞表达共刺激分子CD86,能促进健康人单个核细胞的增殖。
This study was aimed to elucidate the expression of costimulatory molecule CD80 and CD86 in HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction.Acute myelocytic leukemia cell line HL-60 and chronic myelocytic leukemia cell line K562 were cultured.The viability of the cells was measured by flow cytometry.Proteasome inhibitor MG132 at the concentrations of 2 or 3 μmol/L was used to stimulate the HL-60 cell cultured for 24 h and 48 h respectively,and the Annexin V/7-AAD staining and flow cytomotry were used to detect the apoptosis of the HL-60 cells.HL-60 and K562 cells were treated with 1 μmol/L MG132 for 24 h and 48 h respectively,then CD80 and CD86 antibodies were added,finally the expression of CD80 and CD86 was analysed by flow cytomery.The mRNA expression of CD86 in the HL-60 cells treated with 1 μmol/L MG132 was detected by RT-PCR.HL-60 and K562 cells were treated by 1 μmol/L MG132 and then underwent irradiation of 75 Gy 60Co to kill the cells with their antigenicity preserved.Peripheral blood mononuclear cells （PBMNCs） of healthy volunteers,as reactive cells,were isolated and inoculated into the 60Co irradiated HL-60 cells of different concentrations,as stimulating cells,CCK-8 was added and then the A value of absorbance was measured at the wave length of 450 nm in an enzyme labeling instrument.The results showed that the cell viability of the HL-60 cells treated with 1 μmol/L MG132 for 24 h an d 48 h was 92.95% and 85.87% respectively.The apoptotic rates of the HL-60 cells treated with MG132 increased in dose-and time-dependent manner.High-concentration of MG132 directly killed HL-60 cells.Before MG132 treatment K562 cells did not express CD86,but the CD86 expression of the HL-60 cells was up-regulated time-dependently after MG132 treatment （P 〈 0.01）.The mRNA expression of CD86 in the HL-60 treated with MG132 was up-regulated time-dependently （P 〈 0.01）.CCK-8 test showed that the proliferation level of PBMNC gradually increased along
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2014年第5期1251-1255,共5页
Journal of Experimental Hematology
基金
supported by the grants from The Special Science and Research Foundation of Wuhan University of Science and Technology(zx0802)
the Youth and Middle-Age Scientist Science and Research Foundation of the Affiliated Tianyou Hospital,Wuhan University of Science and Technology(xq2008001)
the Foundation of Hubei Provincial Department of Education(530026)
the Natural Science Foundation of Jiangxi Province(0640190)
