| Objective:In multiple myeloma(MM),bone marrow microenvironment plays a important role for the survival,drug resistence and progression of MM cells.The levels of many inflammatory factors were dysregulated,and which were involved the pathogenesis of MM cells.However,It is fully elucidated that how inflammatory factors affect indirectedly on MM cells though BMMSCs in the bone marrow microenvironment.In this research,we investigate the effect of interleukin-1?(IL-1?)or tumor necrosis factor(TNF-?)-primed bone marrow derived mesenchymal stem cells(BMMSCs)on the DOX resistance of myeloma cell lines H929..Methods1.The proliferation of multiple myeloma cell lines H929 or BMMSCs treated with different concentrations of doxycycline(DOX)for 1,2 or 3 day was first assayed by the CCK8 assay.Then CCK8 assay was used to detect the proliferation of H929 when co-cultured with IL-1? or TNF-?-treated BMMSCs or control BMMSCs under DOX treatment.All above mentioned data were collected,and statistically analyzed.2.Real-time PCR was employed to measure the mRNA expression of VCAM-1 and BAFF in BMMSCs treated by the either inflammatory factors.Three groups were set up in the research: 1)group A: BMMSCs without any treatment as control;2)group B: IL-1? treatment on BMMSCs for 1 day;3)group C: TNF-? treatment on BMMSCs for 1 day.The mRNA expression of the three groups were measured by Real-time PCR,and further statistically analyzed.3.Flow Cytometry(FCM)was used to determine the percentage of VCAM-1 positive BMMSCs treated by the either inflammatory factors.Besides,apoptotic status of H929 which co-cultured with pulsed IL-1? or TNF-? treated BMMSCs under DOX was also assayed by FCM.All above mentioned data were collected,and statistically analyzed.4.Western blot was applied for the detection of p-Erk1/2 of H929 cells treated by DOX,or H929 cells co-cultured with pulsed IL-1? or TNF-? treated BMMSCs or control BMMSCs under DOX treatment.All above mentioned data were collected,and statistically analyzed.Results:1.The morphology of BMMSCs cultured in vitro was spindle-shaped.There was no obvious difference in morphology among these groups of BMMSCs,whether BMMSCs were treated by IL-1? or TNF-? and co-cultured by H929.The morphology of H929 cells cultured in vitro was no different whether co-cultured with BMMSCs.2.The concentrations of DOX for this study were identified as 5 and 10?g/ml on the basis of the cell proliferative curve of H929 treated by different concentrations of doxycycline(DOX)for 1day,2 day,or 3 day.The growth of BMMSCs was not obviously influenced by selected concentrations of DOX treatment(P>0.05).The inhibitory effect on H929 by co-culture with IL-1? or TNF-? treated BMMSCs under selected concentrations of DOX was patially reversed(p1 <0.05,p2 <0.05).3.Comparing with control,the mRNA expression level of VCAM-1 was significantly up-regulated in group b and c(P<0.05);but there is no significant difference of the mRNA expression level of BAFF among three groups.4.Comparing with nontreatment BMMSCs,the percentage of BMMSCs with positive VCAM-1 was increased in the group of BMMSCs treated by IL-1? or TNF-?(P<0.05).And the positive rate of VCAM-1 in the group of TNF-? is higher than IL-1? one(P<0.05).The apoptotic rate of H929 co-cultured with BMMSCs was lower than non-cocultured H929 cells treated by 5?g/ml DOX.;but this phenomenon is insignificant under 10?g/ml DOX treatment.The apoptotic rate of H929 when co-cultured with IL-1? or TNF-? treated BMMSCs under DOX treatment was relatively low than the one without inflammatory factors treatment(P<0.05).And the apoptotic rate in TNF-? group is lower than IL-1? group.5.DOX suppress the expression of p-Erk1/2 in H929(P<0.5).The expression of p-Erk1/2 in H929 when co-cultured with IL-1? or TNF-? treated BMMSCs under selected concentrations of DOX was higher,compared to the one without inflammatory factors treatment(P<0.05).Conclusion:Inflammatory factors such as IL-1? and TNF-? treated BMMSCs present the capacity of inhibiting apoptosis of H929 cells induced by DOX.The mechanism of drug resistance effects maybe relate to Mek/Erk pathway and the adhesion molecule of VCAM-1. |
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