The Research Of The Role Of GJIC Composed By Cx43in The Interaction Between Multiple Myeloma Stem Cells And Microenvironment And The Mechanism Study

Part I Title:Isolation of multiple myeloma stem cells and the biological characteristics analysisObjective:To compare the efficacy of different methods in separation of myeloma stem-like cells and to investigate the biological characteristics of myeloma stem-like cells isolated from MM cell lines and primary MM samples.Methods:The magnetic activated cell sorting(MACS) was employed to isolate CD138" cells from6patients with multiple myeloma(MM); Fluorescence activated cell sorter (FACS) was used to detect the porpoortion of SP cells in4kinds of myeloma cell lines (RPMI8226, XG-4, U266, XG-7) and the SP cells of PRM8226were sorted with this assay.To enrich stem cells,the PRIM8226were cultured serum-free medium (SFM) with multiple growth factors such as……The cells proliferation were determined by the growth curve and MTT test.The proportaion of SP cells and the cell cycle were analyzed by FACS. The ability colony-formation in vitro was compared with those in controls in terms of colony forming assay. The expression of c-myc、KIF4、SCX2、OCT4was tested by RT-PCR.The oncogenicity of the different SP cells was analyzed by SCID mouse transplantation tumor experiment in vivo. Seeded subcutaneouslyResults:We have isolated successfully the CD138-MM cells from fresh specimens of6patients with MM, the ratios of the SP cell were:1.42%,1.64%,1.37%,1.54%,1.71%,0.84%. The ratios of SP cell in4kinds of myeloma cell lines were1.78+0.89%(RPMI8226),0.825±0.53%(U266),0.082±0.022%(XG-4) and0.177±0.05%(XG-7) and the SP cells of PRIM8226were sorted successfully with FACs.There are no difference among the proliferation ability of CD138-cells、SP cells and SFM cells. But our data demonstrated that SP subpopulations contain more stem-like cell and the proportions of GO/G1phase were30.41±1.3%(CD138),44.34±1.7%(SP cells) and36.15±1.1%(SFM cells) respectively; The ability of proliferation,colony-forming in vitro, migratory and invasive of SP cells were significantly higher than those of NSP cells (P<0.05). The expression of c-myc, KIF4, SOX2and OCT4were no significant difference among those cells. The SCID mouse transplantation tumor experiment showed that the oncogenicity of SP cells was higher markedly than that of NSP cells.Conclusion:The MM cell lines and the fresh MM bone marrow samples contain a small amount of side population cells, and MACS、FACS、SFM can be used for enriching tumor stem-like cell subgroups. Part II Title:The role of GJIC composed by Cx43in support survival of MM stem-like cells in the bone marrow microenvironment and its possible mechanismObjective:To study the different sources of bone marrow mesenchymal stem cells on myeloma stem-like cell survival and drug resistance and explore the possible role of CX43in it.Methods:cultivate sources-MSCs (MM) patients and normal contrast between mesenchymal stem cells (ND-MSCs), and flow cytometry separation RPMI8226cells or SP cells establish co-culture system directly. Rt-pcr and Western Blot detection respectively before and after co-culture cells BMMSCs and SP/MP CX43in transcription and the expression of protein level, in co-culture system to join channel blocker18alpha glycyrrhetinic acid (alpha GA) by growth curve and determined by MTT detection respectively before and after its appreciation;Flow cytometry analysis of SP cells proportion and the cell cycle;Colony forming experiment testing the clone forming ability;Rt-pcr detection classic symbol expression stem cells.Western Blot detection of Cx43phosphorylation state change and the change of IKKB/NFkB downstream signaling pathways, after joining signaling pathway inhibitor detecting changes in the signal path.Results:We successful separation cultivate sources of patients and normal controls MSC, flow testing the cell phenotype, flow cytometry sorting success SP cells.Patient’s source of MSC in transcription and expression of Cx43protein level higher than that of normal control MSC, SP cells in transcription and expression of Cx43protein level is lower than the MP cell.Directly trained after1h of CX43in BMMSCs and SP cells expressing short hike, express no change after24h. Compared with normal control ND-MSCs trained with the patient’s source of BMMSCs directly after co-culture with8226rising rates of SP cells, increase the proliferation, mediated the BTZ resistant, MM cells of boron for mi cells apoptosis induced by sensitive, with20nmol/L BTZ after24h the apoptotic cells of25.9%plus or minus0.86%, significant difference compared with the controls (6.5%+/-0.2%; p<0.01);By training with BMMSCs directly after24h, boron for the apoptosis induced by microphones decreased significantly, only10.9%plus or minus0.21%(p<0.01), while in the co-culture system add25ummol/L alpha GA, cultivate the apoptotic cells after24h of19.6%plus or minus0.7%(p<0.05), confirmed that the BM-MSCs can to support their growth, not only can be protect RPMI8226cells from antitumor drugs, and channel connection between functional block cells can be partially recovered after the MM cell sensitivity to the boron for zc microphones, improve the proportion of cells in G0, increase the rate of colony formation and nude mouse tumorigenicity, training results have significant difference with normal control MSC.Application channel blocker patients after MSC effect on promoting proliferation of8226cells, on the BTZ reduce resistance, reduce colony formation rate, tumorigenicity.After blocking IKkB/NFkB downstream signaling pathways, channel blocker effect weakened.Conclusion:patients with sources of BMMSCs has better increase myeloma stem cell proliferation, mediated the BTZ resistance, improve the proportion of cells in G0, increase the ability of colony formation rate and nude mouse tumorigenicity, and is closely related to the GJIC function. Cx43/NFkB signaling pathway may be one of its mechanisms. Part III Title:To study the role of GJIC composed by Cx43in the interaction between MM stem cells and microevrimentObjective:To observe the multiple myeloma cells induced ectomesenchymal stem cell microenvironment remodeling process of EMT/MET change and composed of Cx43connected protein GJIC role in this process.Methods:We using flow cytometry instrument separation of multiple myeloma cell lines PRIM8226SP cells, and ectomesenchymal stem cells from normal controls (ND MSCs) and multiple myeloma patients between mesenchymal stem cells (MSCs) MM and human umbilical vein endothelial cells (HUVEC) after training, inverted phase contrast microscope observation of cell morphology change, join channel inhibitors in co-culture system after18alpha glycyrrhetinic acid (ga), cell immunofluorescence staining to observe the cell surface E-calcium mucins, alpha SMA protein expression, WesternBlot methods E-calcium mucins, alpha SMA protein expression changes, and downstream the change of ERK/MAPK signal pathway.Results:We successfully sorting PRIM8226SP cells, and ectomesenchymal stem cells from normal controls (ND MSCs) and multiple myeloma patients between mesenchymal stem cells (MSCs) MM and human umbilical vein endothelial cells (HUVEC) directly after trained for a long time, ND-MSC cells form did not change significantly, while the MM-MSC cells appeared a group of small, accelerated the proliferation, cell migration, cell immunofluorescence staining method for observation and WesternBlot detection E-calcium sticky protein expression and alpha SMA protein expression down;Long-term after co-culture with HUVEC by pebbles sample to spindle cell transformation, cell proliferation, migration, WesternBlot methods E-cut and alpha SMA calcium sticky protein expression protein expression.Conclusion:Multiple myeloma cells induced by microenvironment remodeling, reciprocal transformation between endothelial cells play an important role in it.