| Background Acute myeloid leukemia(AML)is lack of specific therapeutic drugs currently,especially in children with rapid progress and high mortality.Even in patients who achieve remission with chemotherapy relapse is common and occurs from minimal residual disease sequestered in protective niches in the bone marrow microenvironment.In addition,AML cells exhibit a high level of spontaneous apoptosis when cultured in vitro but have a prolonged survival time in vivo,indicating that the tissue microenvironment plays a critical role in promoting AML cell survival.IL-8 is a kind of multi-cellular pro-inflammatory factor,which is highly expressed in many tumor patients,and closely related to the occurrence and development of tumor.It has been reported that bone marrow stromal cells can secrete a variety of cytokines,including IL-8.It is rarely reported in our country whether bone marrow stromal cells secrete IL-8 and IL-8 promote the proliferation of leukemic cells.In this study leukemia cell line(HL-60)was used as a model,we separated bone marrow stromal cells from children with AML,and then investigate whether these bone marrow stromal cells could promote leukemia cell line(HL-60)proliferation by secreting of IL-8.Our work will clarify the role of bone marrow stromal microenvironment on leukemia,which provide a theoretical basis for clinical immunotherapy in children with AML.Objective In this study leukemia cell line(HL-60)was used as a model,we separated bone marrow stromal cells from children with AML,and then investigate whether these bone marrow stromal cells could promote leukemia cell line(HL-60)proliferation by secreting of IL-8.Our work will clarify the role of bone marrow stromal microenvironment on leukemia,which provide a theoretical basis for clinical immunotherapy in children with AML.Methods1.Detect the proliferation of HL-60 cells stimulated by IL-8: HL-60 cells were cultured with 1ng/ml、2ng/ml、4ng/ml、8ng/ml IL-8,and after 24 h,the proliferation of HL-60 cells was detected by MTT.2.Select bone marrow stromal cells from 2 children with AML by density gradient centrifugation.3.Bone marrow stromal cells were co-cultured with HL-60,and the content of IL-8in supernatant was detected by ELISA assay.4.Bone marrow stromal cells were co-cultured with HL-60,the proliferation of HL-60 cells was detected by MTT assay,and the correlation between the proliferation of HL-60 and the content of IL-8 was analyzed.5.Bone marrow stromal cells were co-cultured with HL-60,IL-8 antibody was added to neutralize IL-8 in the supernatant,and ther the apoptosis of HL-60 cells was detected by flow cytometry.6.Bone marrow stromal cells were co cultured with HL-60,IL-8 antibody was added to neutralize IL-8 in the supernatant,and the expression of apoptosis protein Bax was detected by immunofluorescence and confocal laser scanning confocal microscopy.7.Statistical methods: The measurement data to the number of addition and subtraction standard deviation(means±s),the difference between the two groups using t test.P<0.05 means statistically significant.Results1.The proliferation of HL-60 cells stimulated by different concentration of IL-8:1ng/ml,2ng/ml,4ng/ml,8ng/ml concentration of IL-8 stimulated HL-60 cells,when the concentration of IL-8 is less than or equal to 4 ng/ml,the proliferation of HL-60 cells increased with the increase of IL-8 concentration;when IL-8 concentration continues to increase,HL-60 cell proliferation reached the plateau,and didn’t increase obviously anymore.2.BM-MCS cells secreted IL-8: BM-MSC and HL-60 cells were co-cultured,IL-8concentration in supernatant detected by ELISA was(3.83±2.22)ng/ml;in the control group,the IL-8 concentration was(0.68±0.11)ng/ml,the former was significantly higher than that of the latter,and there was significant difference.3.BM-MCS cells secreted IL-8 to stimulate the proliferation of HL-60 cells: BM-MSC and HL-60 cells were co-cultured,the OD value of HL-60 cell by detected by MTT was(15.16±2.03);in the control group,the OD value of HL-60 cell was(10.62±0.90),the former was significantly higher than that of the latter,and there was significant difference.There was a positive correlation between the content of IL-8 and the proliferation of HL-60 cells(R2=0.7673;P=0.0002).4.IL-8 antibodies promoted HL-60 cells apoptosis in the co-culture system:BM-MSC and HL-60 cells were co-cultured and IL-8 antibody was added in the system to neutralize IL-8.Then HL-60 cell apoptosis rate detected by flow cytometry were(8.41±1.70)%;with no IL-8 antibody added,the HL-60 cells apoptosis rate was(2.00±0.59)%,the former was significantly higher than that of the latter,and there was significant difference.5.IL-8 antibodies promoted the bax expression of HL-60 cell in the co-culture system: the bax expression of HL-60 in the co-culture system added IL-8 antibody was higher,which showed a big red granular;without IL-8 being added,the bax expression of HL-60 was lower,which showed a particulate or dust-like.Conclusions1.IL-8 could stimulate the proliferation of leukemia cells(HL-60),and the proliferation rate was dependent of concentration.2.BM-MSC cells from children with AML could secrete IL-8,which could stimulate the proliferation of HL-60 cells,and the proliferation rate was positively correlated with IL-8 concentration.3.IL-8 antibody neutralizing IL-8 secreted by BM-MSC cells in children with AML which could promote the apoptosis of HL-60 cells and the expression of bax. |
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