The Effects Of The Mesenchymal Stem Cells On The Proliferation Of Multiple Myeloma Cells In Vitro And In Vivo

Multiple myeloma (MM) is a malignant plasma cell disorder. It is characterized by heterogeneous anemia, bone disease, renal impairment, hypercalcaemia and infections due to abnormal accumulation of malignant plasma cells in the marrow microenvironment. Multiple myeloma is still incurable and accounts for approximately 10%-15% of human hematopoietic malignancies.Many researches have shown that marrow microenvironment play an important role in the proliferation of human myeloma cells and drug tolerance.【Objective】To research the effects of the mesenchyma stem cells (MSCs) on the proliferation of multiple myeloma cells.Establish plasmacytoma model to determine the effects of the mesenchymal stem cells on the proliferation of multiple myeloma cells in vivo.【Methods】MSCs from fetal bone marrow were isolated and cultured. Direct immunofluorescence assay was employed to analyze the phenotype of those cells by using flow cytometry . Interleukin-6 (IL-6)–dependent human MM cell line XG-7 were cocultured with the MSCs.Before and after coculture,the apoptosis,autophagy and cell cycle of XG-7 were detected by means of annexin V / PI double staining, MDC staining , PI staining. To confirm the presence of gap junctions intercellular communication (GJIC) between cocultured MSCs and XG-7, we use both calcein AM and DiI to labele XG-7 cells. Transwell inserts were used to separate the two kinds of cells in adjacent and remote cocultures, The proliferation of XG-7 cell line was observed in the two different coculture ways. Then we establish plasmacytoma model in there ways to determine the effects of the MSCs on the growth of multiple myeloma cells in vivo: Subcutaneously transplantation of 1cm long segment of 16-18 weeks old human fetal thigh or tibia bone into BALB/c-nu mice to develope a novel model of BALB/c-nu-hu chimera, and three weeks later XG-7 cells were inoculated into the human fetal bone to establish plasmacytoma; the mixture of MSCs and XG-7 cells was injected directerly into subcutaneous of BALB/c-nu mice in the second group; In the third group we injected sigle XG-7 cells into subcutaeous BALB/c-nu mice. 40 days later,human fetal bone implanted as well as the tumor were taken out respectively, stained by hematoxylin and eosin (H&E) and monoclonal mouse anti-human CD34,CD59,CD138 and VEGF followed by morphological examination. Take the X-ray film of mouse model at the start and the end of the experiment.【Results】After cocultured with MSCs, XG-7 cells enhanced the resistance of apoptosis and autophagy, at the same time its proliferation was increased. From fluorescence microscope we observed GJIC between cocultured MSCs and XG-7. Inhibit the GJIC by remote cocultures could step down the growth of XG-7; Fetal bone can survive in the subcutaneous site of BALB/c nude mice;After injection the mixture of the two cells, or injection XG-7 cell into human fetal bone in BALB/c-nu-hu chimera, tumor grown fast and characterized by typical plasmacytoma.And the resorption of the human bones can slso be observed in BALB/c-nu-hu model.But no evidence shows that the tumor was growing in BALB/c-nu mice with single XG-7 cells injection.【Conclusion】MSCs could protect XG-7 cells from apoptosis and autophagy, and obviosely promote the proliferation of XG-7 cells. GJIC dose existence between cocultured MSCs and XG-7, and it may participate the promotion of growth .The BALB/c-nu-hu chimeric model is a novel vector for researching hominine hematopoiesis and bone based on animal. The MM cell can growth successfully based on the BALB/c-nu-hu chimera or subcutaneous injection of mixture cells.