Studies On The Actions Of UPA System And BMSCs In Bone Marrow Microenvironment

Objective: To evaluate the changes about the components of uPA in BMSCs in the microenvironment of leukemia.To establish the co-culture models of BMSCs-K562 and HS-5-K562 and evaluate the changes about the components of uPA in both models.To evaluate the changes about the components of uPA in BMSCs and the co-cultured cells with amiloride incubation.Methods: The mRNA expressions of uPA,uPAR,PAI-1,MMP-9 and VEGF in BMSCs,HS-5 and them in co-culture models were detected by real-time quantitative PCR.MTT method was used to evaluate the inhibition of amiloride to BMSCs.The protein content of uPA,suPAR and PAI-1 in the supernatants of BMSCs,were detected by BMSCs,K562 and HS-5-K562.Results:(1)Compared with HS-5,the expressions of uPA mRNA in T-ALL and M1 were higher;the mRNA expressions of PAI-1 in T-ALL,B-ALL,M5 and M1 were higher;the expression of VEGF mRNA in M1 was higher;the mRNA expressions of uPAR in T-ALL,B-ALL and M5 were higher(P < 0.05).(2)Compared to control,in BMSCs-K562 co-culture model,the mRNA expressions of uPA,uPAR,PAI-1,MMP-9 and VEGF in BMSCs and K562 were much higher after 3 or 4 days culture(P < 0.05),the results are time-dependent;compared to control,in HS-5-K562 co-culture model,the mRNA expressions of uPA,PAI-1 and VEGF in HS-5 were much higher after 3 or 4 days culture(P < 0.05),the results are time-dependent;compared BMSCs-K562 with HS-5-K562 co-culture model,the expressions of mRNA in BMSCs and HS-5 were both time-dependent,but the expressions in BMSCs were higher than that of HS-5.(3)The inhibition rate of amiloride to BMSCs was time-dependent and dose-dependent.After 4 hours incubation with amiloride,the mRNA expressions of uPAR and PAI-1 in BMSCs were lower.After 13 hours incubation with amiloride,in BMSCs,the mRNA expression of uPA was lower and the mRNA expression of MMP-9 was higher;in K562,the mRNA expressions of uPA,uPAR and MMP-9 were lower;in HS-5,the mRNA expression of PAI-1 was lower(P < 0.05).After the incubation with amiloride in HS-5-K562 co-culture model,the mRNA expressions of all the components of uPA system were lower;especially,the mRNA expression of VEGF in K562 was much lower(P < 0.05).(4)Compared with HS-5,the content of uPA and PAI-1 in the supernatants of BMSCs were higher.In BMSCs-K562 and HS-5-K562 co-culture models,the content of uPA in the supernatants was higher than that in mono-cultured BMSCs or HS-5.After 8 hours’ and 13 hours’ incubation with amiloride,the content of PAI-1 in the supernatants of BMSCs was much decreased(P < 0.05).Conclusion:(1)in BMSCs-K562 co-culture models,the expressions of uPA,uPAR,PAI-1,MMP-9 and VEGF in BMSCs were increased.Only the expression of uPA in K562 was increased,but was weaker than that in BMSCs.(2)The expressions of uPA,uPAR and PAI-1 in BMSCs were inhibited by amiloride,but the expression of MMP-9 was increased.Amiloride could inhibit the invasion and progression of leukemia through decreasing the expression of VEGF.