| Objective To investigate the effect and mechanism of allogeneic bone marrow mesenchymal stem cells(BMMSCs)transplantation on bone mass in lupus mice and patients.Methods(1)The urine from mice of 8,12,16,and 20 weeks old were reserved by metabolic cages.(2)Experimental design.C57BL/6:five WT C57BL/6 mice were used as normal control.12w PBS:MRL/lpr mice injected with PBS at 12-week old.12w MSC:MRL/lpr mice injected with BMMSCs at 12 weeks old.12 16w PBS:MRL/lpr mice injected with PBS at 12,16 weeks old respectively.12 16w MSC:MRL/lpr mice injected with BMMSCs at 12,16 weeks old respectively.The femurs were isolated,then HE staining was used to observe the trabecular bone and the expression of P-Smadl/5/8 protein was detected by immunohistochemistry.(3)Bone marrow mesenchymal stem cells(BMMSCs)were isolated by the whole bone marrow culture and adherence screening methods.The capacity of osteogenic differentiation of BMMSCs was analyzed by Alkaline phosphatase and alizarin red staining..The mRNA level of Runx2,NF-κB and BMP/Smad signaling pathways were detected by RT-PCR.(4)The protein levels of bone morphogenetic protein-2(BMP-2),osteocalcin(OCN),nuclear factor kappa B receptor activator ligand(RANKL)and osteoprotegerin(OPG)were detected by ELISA,then the ratio of OPG/RANKL were calculated.(5)The serum levels of BMP-2,OCN,OPG,RANKL,total type Ⅰ procollagen amino-terminal peptides((t-PINP)and type Ⅰ collagen carboxy-terminal peptides(P-CTX)from patients with systemic lupus erythmatousus(SLE)were measured and compared before and after UC-MSCs transplantation.(6)The peripheral blood mononuclear cells of SLE patients were isolated and the mRNA levels of Runx2 and several genes of NF-κB and BMP/Smad signaling pathways were measured by RT-PCR.Results(1)The amount of 24h proteinuria of MRL/lpr mice was higher than that of normal control group.After BMMSCs transplantation,the amount of proteinuria decreased.(P<0.05).Moreover,proteinuria in 12 16w MSC group was lower than that in 12w MSC group.(2)The trabecular of bone tissue of MRL/lpr mice showed disorder and the trabecular bone area of the lupus mice were decreased(P<0.05).After BMMSCs transplantation,the area of trabecular bone were increased(P<0.05).while there was no difference between the two groups of transplantation..Bone tissue immunohistochemistry showed that the number of P-Smadl 1/5/8 positive osteoblasts was significantly higher in lupus mice,and increased after transplantation,but still less than normal control mice.(3)ALP activity and mineralization nodules of BMMSCs in MRL/lpr mice were attenuated(P<0.05).The mRNA levels of Runx2(P<0.05).BMP2 and Smadl(P<0.01)in lupus mice were lower).,while that of TNF-a,NF-KBp50 and NF-κBp65 were found higher in lupus mice(P<0.01);After BMMSCs transplantation,the genes expression of TNF-a,NF-KBp50 and NF-KBp65 dereased,meanwhile the gene expression of BMP2 increased(P<0.05).(4)The serum levels of BMP-2,OCN and OPG in MRL/lpr mice were foud significantly lower(P<0.001),and increased after BMMSCs transplantation(P<0.01)The level of RANKL was much higher than those in normal controls(P<0.001)and sharply decreased after the transplantation(P<0.01).(5)The serum levels of BMP-2 and t-PINP in SLE patients increased after UC-MSCs transplantation(P<0.05)and the serum of RANKL decreased(P<0.05).There were no difference in the serum levels of β-CTX,OCN and OPG after the transplantation in SLE patients.(6)After transplantation,the mRNA levels of TNF-a,NF-KBp50 and NF-KBp65 ofPBMC from SLE patients decreased,and the levels of Runx2,BMP-2,Smadl,Smad5 and Smad8 did not not change.ConclusionsWe found osteoporosis in MRL/lpr mice.The capacity of osteogenic differentiation of BMMSCs from MRL/lpr mice decreased,and the BMMSCs transplantation could partly improve the metabolic indexes of lupus mice and SLE patients.Meanwhile the transplantation could improve the osteoporosis in MRL/lpr mice BMMSCs transplantation may improve the bone metabolism by BMP and NF-κB signaling pathways. |
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