The Involvement Of Nuclear Factor-кb Signaling Pathway In The Cell Proliferation/apoptosis Of Human Mesenchymal Stem Cells

Direction: To investigate the effect of nuclear factorкB signaling pathway in the human mesenchymal stem cells whether it is relative to the regulation about the cell proliferation and apoptosis.Material and methods :(1)The way to gain the hMSCs: collect the bone marrow during the total hip replacement operation,then adhere it in the culture vessels,with cultural nutrition.Culture the cells until it grew so many as to be flushed and purified by flushing.Then observe it under light microscope. (2)Cells identification: use flow-cytometric assay to test the surface molecules to identify the hMSCs.(3)Interention and test:culture the cells until its number was large enough to be added lipopolysaccharids to up-regulate the expression of the protein p65 in the NF-кB signaling pathway.Use the Western blotting to test the expression of the protein p65.(4) Detect the expression of PARP and PCNA,which might be related to cell proliferation or apoptosis.(5)Use the MTT to detect the proliferation rate of the cells with different medicine mentioned before and observe the differences.(6)Mark the cells with PI,then test the apoptosis rate of the cells with different intervene by flow-cytometric assay.Results:(1)The mesenchymal stem cells can be separated from the bone marrow got from the total hip replacement,and can be cultured in the lab.It appreared well morphologically. (2)The flow-cytometric assay test showed that the cells cultured can be conformed to be the hMSCs,judged from the surface molecules.(3) The cells purified during the culture treated with the LPS (1ug/ml) and PDTC (25mM) showed in the Werstern Blotting way that with the stimulation from the LPS,the expression of the p65 was up-regulated,which conformed that the NF-кB signaling pathway was stimulated.(4)Other proliferation/apoptosis-associated proteins were diffrent in expression when NF-кB was stimulated. (5) The MTT test showed that with the stimulation,the hMSCs was up-regulated in proliferation in short time.(6)The flow-cytometric assay test suggested that the stimulation of the NF-кB signaling pathway had a lower apoptosis rate.Conclusion:(1)The cells gained from the patients duriong the opertion can be seperated and cultured,and was able to be identifide to be the hMSCs by flow-cytometric assay. (2)With the effect of the LPS,human mesenchymal stem cells expressed p65 in a higher level,which indicated the NF-кB signaling pathway was stimulated.Accordingly, the proliferation /apoptosis associated proteins such as poly ADP-ribose Polymerase (PARP) and the Proliferating Cell Nuclear Antigen(PCNA),had a higher expression,with whose effect the hMSCs had a up-regulated proliferation rate. What's more,the activation of the NF-кB might protect hMSCs against apoptosis.