| Objective:To study whether the bone marrow mesenchymal stem cells(BM-MSCs)can promote the multidrug resistance of leukemia,and further to explore the effect of tetrandrine on multidrug resistance mediated by BM-MSCs and its possible mechanism.Methods:(1)Following establishment of a co-culture system of BM-MSCs and K562 cell line,CCK-8 was performed to compare the proliferation of K562 in the co-culture group and the K562 cell culture group after treatment with tetrandrine;(2)Co-culture in Transwell chambers was used to observe the interaction between K562 cell line and BM-MSCs,and to explore the mechanism of drug resistance reversing mediated by tetrandrine;(3)Co-culture group and K562 group were incubated with tetrandrine.ELISA and flow cytometry were used to determine the cytokine concentration in the supernatant(such as CXCL12,IFN,IL-2 and IL-6).The expression of hub genes was verified by Western blot and RT-q PCR at protein and m RNA levels.Results:(1)Based on the CCK-8 assay,it was found that after co-culture with BM-MSCs,inhibition rate of DNR on K562 decreased significantly(P<0.05).After combination with TET(1μmol/L)in the co-culture system,the inhibition rate increased(P<0.05).(2)In the groups using Transwell chambers,it was found that compared with the non-Transwell group,the inhibition rate of DNR monotherapy intervention group increased(P<0.05).In the combination of TET(1μmol/L)intervention groups,the inhibition rate of DNR also increased after using the Transwell chambers(P<0.05).(3)Based on the CBATM,compared to the vehicle control,it was found that the level of IL-6 in co-culture group increased significantly(P<0.05),with an increase of 490.18 times.After adding TET to the co-culture group,the level of IL-6 in the cell culture supernatant decreased significantly(P<0.05),which was reduced by 1.51 time.While there were no significant differences in the concentration levels of IL-2,IFN and CXCL12 in two groups(P>0.05).Among them,the co-culture system had a significant difference in IFN concentration levels(P<0.05)compared with the individual culture group(P<0.05),while no difference of IFN concentration was found in the co-culture group after intervention of TET compared with the non-intervention group(P>0.05).(4)According to the results of RT-q PCR,we found that the transcription levels of MDR1 and STAT3 were upregulated after being cultured with BM-MSCs,and the levels were downregulated after treatment with TET(P<0.05).(5)From the result of Western blot analysis,we found that the level of P-gp increased,and then declined with the additional treatment of TET(P<0.05).Also,the level of p-STAT3/STAT3 in the co-culture group was found to be up-regulated.Conversely,the level of p-STAT3/STAT3 was down-regulated in the combination of TET group(P<0.05).Conclusion:Bone marrow microenvironment did have promoting effects on the drug resistance in leukemia.TET may reverse this kind of effects by inhibiting the expression of P-gp and the IL-6/STAT3 pathway. |
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