Study On The Mechanism Of Aging Bone Marrow Mesenchymal Stem Cell Conditioned Medium Promoting The Malignant Proliferation Of Multiple Myeloma

Objective:Human bone marrow Mesenchymal stem cells(MSCs)were cultured and their aging model was established.The effects of Conditioned medium for mesenchymal stem cells(MSC-CM)on the proliferation of Multiple myeloma(MM)were collected and compared.To further explore the mechanism of aging MSCs on the malignant proliferation of multiple myeloma(MM).Methods:(1)Identification of MSCs and establishment of aging model: Bone marrow blood samples from normal subjects were collected,and mesenchymal stem cells from bone marrow of healthy donors were purified by Ficoll density gradient centrifugation and adherent method.MSCs was induced and cultured with 200μM H2O2 for 2h,then the aging model of MSCs was established.The third generation MSCs were stained with crystal violet for morphology observation,and their surface markers were identified by flow cytometry.Alizarin red,oleored O and toluidine blue staining were used to identify the multidirectional differentiation ability of MSCs in the two groups,and β-galactosidase specific staining was used to identify senescence.MTT colorimetric proliferation method was used to determine the growth curves of young and aged MSCs.Cell proliferation rate was detected by Ed U fluorescence staining.Western blot and real-time PCR were used to verify the expression of senescence related genes at protein and m RNA levels,respectively.(2)the young and the old MSC-CM’s influence on the MM cell lines: collect between 3-5 generations of normal and aging of mesenchymal stem cells in culture medium,after ultrafiltration centrifugal controls ectomesenchymal stem cell conditioned medium for enrichment,the induction of multiple myeloma cell line 48 hours,the proliferation determined by MTT colorimetric method,Ed U fluorescence staining detection MM cell proliferation;Transwell was used to detect cell mobility.Cell apoptosis rate was detected by flow cytometry.(3)The molecular mechanism of MSC-CM-mediated Wnt/β-catenin/Hippo pathway on MM proliferation: Bone marrow blood samples from normal donors and MM patients were collected,mononuclear cells were extracted by Ficoll density gradient centrifugation,and the expression of YAP1 was measured by Western blot.Double antibody sandwich immunoassay(ELISA)was used to detect the content of IL-6 in MSCs concentration medium of the two groups,and Western blot was used to detect the expression of Wnt/β-catenin/Hippo signaling pathway.Verteporfin(VP),the optimal concentration of YAP inhibitor,mediates the down-regulation of the expression of the target gene YAP1,Western-blot detection of the downstream target gene p73 of YAP1 signal pathway 24 h before and after VP treatment.Expression of anti-apoptotic genes Bcl-2 and BTK at protein level.All data were obtained by 3 independent repeated experiments.Data were analyzed by SPSS23.0 and plotted and analyzed by Graphpad Prism 8.3 software.Continuous variable data were expressed as mean ± standard deviation(SD).P < 0.05 was considered as significant difference,and statistical chart was drawn.Results:(1)Ficoll density gradient centrifugation and adherent purification were used to separate MSCs.The aging MSCs model was established by 200μM H2O2 induction.Flow cytometry was used to detect the immunophenotypes of normal and aging MSCs,and the results showed that CD90,CD105 and CD73 were highly expressed in both groups,but CD14,CD34 and CD45 were not expressed.CD11 b and HLA-DR;The MSCs in both groups had osteogenic,adipogenic and chondrogenic differentiation potential.Compared with the young group,the multidirectional differentiation ability of MSCs in the aging group was decreased.β-galactosidase specific staining was used to identify aging cell model.The positive rate of aging MSCs was higher than that of young MSCs.Compared with the young group,the expression of p53,p16 and p21 in the aging group MSCs increased,and the growth rate of aging MSCs slowed down.(2)MM cell lines RPMI8226 and U266 were treated with young MSC-CM and aged MSC-CM.Compared with MM cell lines induced by young MSC-CM,the proliferation and migration rate of aged MSC-CM increased and the apoptosis rate decreased.(3)ELISA results showed that the content of IL-6 in conditioned medium of aging MSCs was higher than that in conditioned medium of young MSCs;Western blot were used to detect the expression of YAP1 in bone marrow mononuclear cells.It was found that the expression of YAP1 in normal donors was higher than that in MM patients.Western blot was used to detect MM cell lines treated with different conditioned media,and it was found that: Compared with young MSC-CM,the Wnt/β-catenin/Hippo pathway was activated in aging MSC-CM treated MM cell lines,and the expression of apoptosis-related genes p73 decreased,while the expression of anti-apoptosis-related genes Bcl-2 and BTK increased.The expression of anti-apoptosis-related genes was increased,β-catenin expression was up-regulated,YAP1 and p73 expression was down-regulated in MM cell line.Conclusions:Aging MSCs conditioned medium has a stronger effect on promoting the malignant proliferation of multiple myeloma,which may activate the Wnt/β-catenin/Hippo pathway,down-regulate YAP1 and promote MM cell proliferation by secreting high levels of IL-6.