| Objective:To investigate the expression level of CXCL12 on the surface of Osteoblasts of MRL/lpr lupus mice and its effect on the early differentiation of B lymphocytes.To observe the effects of BMMSCs infusion on the improvement of disease indicators,CXCL12 expression in bone marrow and CXCR4 expression on B and plasma cell surface in MRL/lpr lupus mice.Methods:(1)The experimental groups were:C57BL/6 mouse group(C57BL/6)and MRL/lpr lupus mouse group(MRL/lpr).The expression levels of complement C3 and immunoglobulin Ig G in the serum of C57BL/6 mice and MRL/lpr lupus mice were detected by ELISA.Then hematoxylin-eosin staining was used to observe the pathological changes of the kidney.The expression of CXCL12 in bone marrow of C57BL/6 and MRL/lpr lupus mice was detected by q RT-PCR.Flow cytometry was used to test the ability of C57BL/6 and MRL/lpr mice bone marrow cells to turn into pre-pro-B,pro-B,pro-B.Differentiation of bone marrow cells into pre-B in C57BL/6 and MRL/lpr lupus mice was detected by CFU.The expression levels of CXCR4 in B and plasma cells of bone marrow were detected by flow cytometry.(2)The experimental groups of co-culture system were as follows:1.OB of C57BL/6mice was used as support cells,and HSCs of C57BL/6 mice were added(C57 OB+C57HSC group);2.OB from MRL/lpr lupus mice was used as supporting cells and added into HSCs of C57BL/6 mice(MRL OB+C57 HSC group);3.OB of C57BL/6 mice was used as supporting cells and added into HSCs of MRL/lpr lupus mice(C57 OB+MRL HSC group);4.OB cells of MRL/lpr lupus mice was used as supporting cells and added into HSCs of MRL/lpr lupus mice(MRL OB+MRL HSC group).The cranial OB of MRL/lpr lupus mice was collected and identified with alizarin red staining after one and two weeks of stimulation with osteogenic induction solution.QRT-PCR was used to detect the difference in C57BL/6 mice and MRL/lpr lupus mice cranial OB CXCL12 expression.OB of the two kinds of mice was co-cultured with C57BL/6mice and MRL/lpr lupus mice hematopoietic stem cells for one week,and then using flow cytometry to detect the proportion of CD19~-B220~+and CD19~+B220~+cells,and observe the effect on the early differentiation of HSCs into B cells.(3)The experimental groups were as follows:1.MC3T3-E1 was supporting cell,and HSCs of C7BL/6 mice were added(MC3T3+C57 HSC);2.MC3T3-E1 transfected with sh RNA340 was used as supporting cell,and C57BL/6 mouse HSCs were added(sh340+C57 HSC).After the lentiviral vector is constructed,it is co-transfected with MC3T3-E1 cells to decrease the expression of CXCL12,and then q RT-PCR is used to detect the decrease level.MC3T3-E1 was co-cultured with C57BL/6 mouse hematopoietic stem cells,and then the proportion of CD19~-B220~+and CD19~+B220~+cells was detected by flow cytometry to observe the effect of HSCs on the ability of early differentiation to B cells.(4)Experimental grouping:1.PBS infusion group(PBS);2.2.BMMSCs group(MSC).BMMSCs of C57BL/6 mice cultured to the third generation were infused into MRL/LPR mice via tail vein,and PBS was infused as the control group.They were executed at the end of 20 weeks of age.The peripheral blood supernatant was taken for ELISA to detect the level of immunoactive substances,the kidney was taken for hematoxylin-eosin staining,the bone marrow was taken for q RT-PCR to detect the expression of CXCL12,and the surface of B and plasma cells was detected by flow cytometry.Results:(1)Compared with C57BL/6 mice,serum complement C3 level of MRL/lpr lupus mice was lower and Ig G level was higher;The proportion of bone marrow cells into pre-pro-B,pro-B,pre-B and immature B was lower in MRL/lpr lupus mice.The expression of CXCL12 increased in bone marrow of MRL/lpr lupus mice.The expression of CXCR4on bone marrow B cells and plasma cells was increased in MRL/lpr lupus mice.(2)The expression of CXCL12 was down-regulated in MC3T3-E1 cells transfected with sh RNA340.After one week of direct contact and co-culture with HSCs of C57BL/6mice,the percentages of CD19~-B220~+pre-pro-B and CD19~+B220~+pro-B were decreased by flow cytometry.(3)The expression of CXCL12 in MRL/lpr lupus mouse OB was higher than that in C57BL/6 mouse OB.The proportions of CD19~-B220~+pre-pro-B and CD19~+B220~+pro-B in C57BL/6 HSCs in lupus mouse OB environment were higher than those in normal mouse OB environment.The ratio of CD19~-B220~+pre-pro-B in lupus HSCs in lupus mouse OB environment was higher than that in normal mouse OB.In both the OB environment of C57BL/6 mice and the OB environment of MRL/lpr lupus mice,the HSCs of MRL/lpr lupus mice differentiated into CD19~+B220~+pro-B in a lower proportion than those of C57BL/6 mice.(4)The expression of complement C3 was increased and Ig G was decreased in BMMSCs group compared with PBS group.Kidney HE staining indicated improvement of renal inflammation in lupus mice after infusion of BMMSCs.The expression level of CXCL12 in the bone marrow of the BMMSCs group was lower than that of the PBS group.In addition,the expression rate of CXCR4 on the surface of bone marrow B cells and plasma cells in the BMMSCs group was lower than that in the PBS group.Conclusion:The differentiation ratio of HSCs in bone marrow to immature B cells in MRL/lpr lupus mice decreased,and the expression of CXCL12 and CXCR4 on the surface of B and plasma cells in bone marrow increased.The high expression of CXCL12 in osteoblasts can promote the early differentiation of HSCs into B cells,while the low differentiation of bone marrow B lymphocytes in the immature stage of MRL/lpr lupus mice may be caused by both the defects of HSCs and the bone marrow microenvironment constructed by OB.BMMSCs infusion corrected the expression of CXCL12 in bone marrow and CXCR4 on the surface of plasma cells,and improved the disease activity of MRL/lpr lupus mice. |
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