【作者】 周咏明；

【导师】 黄士昂；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 研究目的研究蛋白酶体抑制剂MG132诱导急性髓性白血病细胞HL-60细胞表达共刺激分子CD80、CD86的信号转导途径,诱导HL-60细胞的凋亡途径及其对混合淋巴细胞反应的作用。研究方法流式细胞仪检测MG132诱导HL-60细胞表达共刺激分子CD80、CD86及细胞活力和细胞凋亡,并检测用蛋白激酶通路抑制剂G?6976、JNK通路抑制剂SP600125、Caspase-8抑制剂Z-IETD-FMK、Caspase-9抑制剂Z-LEHD-FMK、组蛋白去乙酰化酶抑制剂曲古菌素A和p21和p27MAPK通路抑制剂Apigenin阻断蛋白酶体抑制剂MG132蛋白激酶通路、JNK通路、、Caspase-8通路、Caspase-9通路、组蛋白乙酰化酶通路和p21和p27MAPK通路后MG132诱导HL-60细胞CD80与CD86的表达,检测应用Caspase-8抑制剂Z-IETD-FMK和Caspase-9抑制剂Z-LEHD-FMK阻断MG132的Caspase-8和Caspase-9通路后HL60细胞的凋亡;RT-PCR分析蛋白酶体抑制剂MG132诱导HL-60表达共刺激分子CD80和CD86 mRNA的情况,分析阻断蛋白酶体抑制剂MG132蛋白激酶通路、JNK通路、、Caspase-8通路、Caspase-9通路、组蛋白乙酰化酶通路和p21和p27MAPK通路后MG132诱导CD80与CD86 mRNA的表达;Western blot检测MG132诱导HL-60细胞凋亡后,p21、p27和p53蛋白的表达;75Gy照射MG132诱导的HL-60细胞后,用CCK-8检测提高共刺激分子表达后,HL-60细胞对健康人单个核细胞的增殖作用。研究结果小剂量的MG132上调HL-60细胞表达CD86,不诱导CD80的表达,应用p21和p27MAPK通路抑制剂Apigenin阻断蛋白酶体抑制剂MG132诱导HL-60细胞表达CD86的p21和p27MAPK通路后,CD86的表达明显减少,CD80变化不明显,表明MG132诱导HL-60细胞表达共刺激分子CD86可能通过p21和p27MAPK途径;而应用蛋白激酶通路抑制剂G?6976、JNK通路抑制剂SP600125、Caspase-8抑制剂Z-IETD-FMK、Caspase-9抑制剂Z-LEHD-FMK、组蛋白去乙酰化酶抑制剂曲古菌素A阻断MG132诱导HL-60细胞表达共刺激分子CD86的蛋白激酶通路、JNK通路、、Caspase-8通路、Caspase-9通路、组蛋白乙酰化酶通路后,CD86无明显变化。大剂量的MG132对HL-60细胞有直接杀灭作用,可诱导HL-60细胞的凋亡,应用Caspase-8抑制剂Z-IETD-FMK、Caspase-9-LEHD-FMK抑制剂阻断MG132诱导HL-60细胞的Caspase-8通路、Caspase-9通路后,细胞凋亡无明显变化,表明MG132诱导HL-60的凋亡可能不通过Caspase-8通路、Caspase-9通路。Western blot检测MG132诱导HL-60细胞凋亡后p21、p27、p53蛋白的表达,p21蛋白、p27蛋白在MG132作用HL-60细胞后表达升高,p53蛋白变化不明显。用MG132提高HL-60细胞表达共刺激分子CD86后,对健康人单个核细胞起增殖作用。研究结论小剂量的蛋白酶体抑制剂MG132诱导HL-60细胞表达共刺激分子CD86,其诱导CD86表达的途径可能与p21和p27MAPK途径有关,而与蛋白激酶通路、JNK通路、、Caspase-8通路、Caspase-9通路、组蛋白乙酰化酶通路无关。大剂量的MG132诱导HL-60细胞凋亡,对HL-60细胞有直接杀灭作用,MG132诱导的HL-60细胞凋亡不通过caqspase-8和caspase-9途径,p21蛋白、p27蛋白可能参与了MG132诱导的HL-60细胞凋亡,p53蛋白的表达无明显变化。小剂量MG132促进对健康人单个核细胞的增殖作用,可提高机体的免疫性。更多还原

【Abstract】 Objective To investigate the role of proteasome inhibitors MG132 in the inducing costimulatory molecules CD80 and CD86 expression, signal transduced pathway, apoptosis and pathway in leukemic cells and its effect of 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 examined the viability of the cells. MG132, a proteasome inhibitors, of the concentrations of 2 or 3μmol/L was added into the culture fluid of HL-60 cells for 24h and 48h respectively and then annexinⅤ/7-AAD staining and FC were used to detect the apoptosis of the apoptosis of the cells. HL-60 and K562 cells treated with 1μmol/L MG132 for 24h and 48h 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 1μmol/L MG132 was examined by RT-PCR. Protein kinase inhibitor G?6976,JNK inhibitor SP600125, caspase-8 inhibitor Z-IETD-FMK, caspase-9 inhibitor Z-LEHD-FMK,histone deacetylase inhibitor Trichostatin A and p21 and p27 MAPK inhibitor Apigenin blocked protein kinase pathway, JNK pathway caspase-8 pathway, caspase-9 pathway, histone acetylase pathway and p21 and p27 MAPK pathway in MG-132-inducing the expression of CD86 in HL-60 cells by Flow Cytometer measuring. caspase-8 inhibitor Z-IETD-FMK and caspase-9 inhibitor Z-LEHD-FMK blocked caspase-8 pathway and caspase-9 pathway in MG132-inducing apoptosis in HL-60 cells by Flow Cytometer measuring. The expression of CD86 mRNA analyzed by RT-PCR after Protein kinase inhibitor G?6976, JNK inhibitor SP600125, caspase-8 inhibitor Z-IETD-FMK, caspase-9 inhibitor Z-LEHD-FMK,histone deacetylase inhibitor Trichostatin A and p21 and p27 MAPK inhibitor Apigenin blocked protein kinase pathway, JNK pathway caspase-8 pathway, caspase-9 pathway, histone acetylase patway and p21 and p27 MAPK pathway in MG132-induced the expression of CD86 in HL-60 cells. HL-60 and K562 cells were treated by 1μmol/L MG132 for 48h 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 K562 cells of different concentrations, as stimulating cells, for 5d, CCK-8, a new agent to detect the cell viability, was added for 4h, and then the A value of absorbance was measured at the wave length of 450 nm of enzyme labeling instrument. Control groups were setn up for all tests.Results The cell viability rates of the HL-60 cell treated with 1μmol/L Mg132 for 24h and 48h were 92.95% and 85.87% respectively. The apoptotic rates 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) treatment with low-dosage MG132. The mRNA expression of CD86 in the HL-60 treated with MG132 was up-regulated time-dependently (P<0.001). The expression of CD80 was unchanged. The expression of CD86 was decreased in HL-60 cells after p21 and p27 MAPK inhibitor Apigenin blocked p21 and p27 MAPK pathway treatment with MG132. It showed that MG132-induced the expression of CD86 may via p21 and p27 MAPK pathway. Low-dose MG132 induced the expression of CD86 in HL-60 cells. No significant changes were observed in MG132-induced the expression of CD86 in HL-60 cells after inhibiting protein kinase pathway, JNK pathway, histone acetylase pathway, caspase-8 pathway and caspase-9 pathway. High-dose MG132 directly killed HL-60 cells and induced apoptosis in HL-60 cells. No significant changes were observed in MG132-induced apoptosis after inhibiting caspase-8 and caspase-9 pathway. The expression of p21 protein and p27 protein increased in MG132-induced apoptosis. The expression of p53 protein was unchanging in MG-132-induced apoptosis in HL-60 cells. CKK-8 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 1×105(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 Low-dose protesome inhibitor MG132 induces the expression of costimulatory molecular CD86 expression in HL-60 cells, and associated with p21 and p27 MAPK pathway but not associated with protein kinase pathway, JNK pathway, histone acetylase patway, caspase-8 pathway and caspase-9 pathway, improves the proliferation of PBMNCs. High-dose MG132 induced apoptosis and directly killed HL-60 cells. MG132-induced apoptosis via caspase-8- and caspase-9-independent pathway. P21 protein and p27 protein was involved in MG132-induced apoptosis in HL-60. No significant changes were observed in MG132-induced the expression of p53 protein in HL-60 cells. Low-dose MG132 improved the proliferation of PBMNCs from healthy volunteers and enhanced immunity.更多还原