Effect Of N-Cadherin On Osteoblates On The Hematopoiesis Impairment In MRL/lpr Lupus Mice

Objective:To test the number of peripheral blood cells,hematopoietic cells of bone marrow and evaluate the extramedullary hematopoiesis of MRL/lpr mice.To investigate the effect of N-Cadherin on bone marrow osteoblasts(OB)from MRL/lpr and C57BL/6mice on the differentiation of hematopoietic stem cells(HSCs).To observe the effect of bone marrow masenchymal stem cells(BMMSCs)intravenous infusion on the hematopoiesis of lupus mice.Methods:1.The number and proportion of peripheral blood,bone marrow and spleen cell lines in MRL/lpr lupus mice and C57BL/6 mice were analyzed by blood cell analyzer and flow cytometry.The number of CFU-pre-B,BFU-E and CFU-GM in bone marrow and spleen of MRL/lpr lupus mice and C57BL/6 mice was detected by methyl cellulose semi-solid colony formation test(CFU).Experimental group: 1.MRL/lpr lupus mice Group(MRL/lpr);2.C57BL/6 mice Group(C57BL/6).2.The BMMSCs of MRL/lpr lupus mice was cultured to the third generation,directed into bone differentiation,and the osteogenesis cells were identified by the Western blot,and the expression of N-Cadherin in various stages of osteoblast differentiation was detected.The expression of osteoblasts N-Cadherin in MRL/lpr lupus mice and C57BL/6 mice was compared.The osteoblasts of MRL/lpr lupus mice and C57BL/6 mice were co-cultured with hematopoietic stem cells in MRL/lpr lupus mice and C57BL/6 mice respectively,and then CFU was used to detect the differentiation ability of hematopoietic stem cells.Co-culture system experimental group: 1.The osteoblasts of C57BL/6 mice were feeder layer cells,and HSCs of C57BL/6 mice were added into the culture medium(C57+HSCs group);2.The osteoblasts of C57BL/6 mice were feeder layer cells,and HSCs of MRL/lpr lupus mice were added into the culture medium(C57+MHSCs group);3.The osteoblasts of MRL/lpr lupus mice were feeder layer cells,and HSCs of C57BL/6 mice were added into the culture medium(MRL+HSCs group);4.Theosteoblasts of MRL/lpr lupus mice were feeder layer cells,and HSCs of MRL/lpr lupus mice were added into the culture medium(MRL+MHSCs group).3.The expression of N-Cadherin was reduced by using lentivirus infection before osteoblast line MC3T3-E1,and the expression level of N-Cadherin was identified by Western Blot.The MC3T3-E1 cells before and after infection were co-cultured with hematopoietic stem cells in MRL/lpr lupus mice and C57BL/6 mice,and the differentiation ability of hematopoietic stem cells was detected by CFU.Co-culture experiment group: 1.MC3T3-E1 for feeding layer cells,culture medium added C57BL/6 mice HSCs(MC3T3+HSCs group);2.MC3T3-E1 for feeding layer cells,culture medium added MRL/lpr lupus mice HSCs(MC3T3+MHSCs group);3.after infection 1070 cells for the feeding layer cells,the culture medium added C57BL/6 mouse HSCs(1070+HSCs group);4.after infection1070 cells for the feeding layer cells,culture medium added MRL/lpr lupus mice HSCs(1070+MHSCs group).4.The BMMSCs of C57BL/6 mice was isolated and cultured by whole bone marrow adherence screening method,and the tail vein infusion was carried out on the16-week-old MRL/lpr lupus mice,and the phosphate buffered saline was injected as the control group.The number and proportion of peripheral blood,bone marrow and spleen cell lines of MRL/lpr lupus mice were analyzed by blood cell analyzer and flow cytometry at 20 weeks old,and the difference of the formation number of hematopoietic progenitor cells in bone marrow and spleen was detected by CFU method.Results:1.Compared with C57BL/6 mice,the proportion of red blood cells,hemoglobin and platelets decreased,T lymphocytes,memory B cells and plasma cells increased in peripheral blood of MRL/lpr lupus mice,and the proportion of total B lymphocytes decreased.In the bone marrow of MRL/lpr lupus mice,the proportion of total B lymphocytes,pro-B cells and pre-pro-B cells decreased,and the proportion of plasma cells and HSCs increased.In the spleen of MRL/lpr lupus mice,the proportion of totalB lymphocytes decreased,the proportion of plasma cells increased,and the proportion of medullary system decreased.2.The differentiation ability of MRL/lpr lupus mice HSCs to pre-B decreased.3.The differentiation of MRL/lpr lupus mice HSCs decreased to B lineage and erythroid lineage with C57BL/6 OB support;The differentiation of C57BL/6 HSCs increased to B lineage and erythroid lineage with MRL/lpr lupus mice OB support;N-Cadherin expression of MRL/lpr lupus mice OB was higher than C57BL/ 6 mice;lentivirus infected MC3T3-E1 pre-osteoblasts,successfully down-regulated the expression of N-Cadherin.After N-Cadherin down-regulation,MC3T3-E1 supported the decrease of HSCs to B lineage and erythroid differentiation.4.MRL/lpr lupus mice were injected into normal mice with BMMSCs,the number of platelets in peripheral blood of receptor mice was improved,the proportion of plasma cells decreased,the proportion of Granulocytes-megakaryocyte line in bone marrow increased,the proportion of T lymphocytes and stem cells decreased,the differentiation of T lymphocyte to B system increased,the proportion of in spleen decreased,and the proportion of stem cells increased.Conclusion:There was abnormal differentiation of B lymphocytes in peripheral blood,bone marrow and spleen hematopoietic abnormalities in MRL/lpr lupus mice.The high expression of N-Cadherin on the surface of OB can promote the differentiation of HSCs to B lineage,and N-Cadherin plays an important role in the differentiation of HSCs in bone marrow niches.BMMSCs infusion had an effect on hematopoietic cells in peripheral blood,bone marrow and spleen of MRL/lpr lupus mice.It is of therapeutic significance for the damage of blood system in MRL/lpr lupus mice.