Effects Of Bone Marrow Mesenchymal Stem Cells N-Cadherin On Bone Formation And Hematopoiesis In MRL/lpr Lupus Mice

Objective:Patients with systemic lupus erythematosus(SLE)with pancytopenia currently lack effective treatment.This project intends to study the effects of bone marrow mesenchymal stem cells BMMSCs N-Cadherin(N-cadherin)on the bone formation and hematopoiesis of MRL / lpr lupus mice,providing a new theoretical basis for SLE pancytopenia and a new target for treatment point.Method:(1)qRT-PCR was used to detect the mRNA expression difference of BMMSCs N-cadherin in C57 BL / 6 mice and MRL / lpr mice;(2)qRT-PCR was used to detect the difference in expression of N-cadherin before and after osteoblast induction of MRL / lpr BMMSCs;(3)qRT-PCR was used to detect the difference of osteogenic indexes(Col1A1,ALP,Run2,OCN)of BMMSCs in C57 BL / 6 mice and MRL / lpr mice;(4)Differences in bone indexes of qRT-PCR between C57 BL / 6 mice and MRL / lpr mice BMMSCs 2 weeks after osteogenic induction;(5)Construction of lentivirus,transfection of cells,down-regulation of N-cadherin expression of MRL / lpr murine BMMSCs,flow screening of GFP green fluorescence positive cells,and identification of low expression groups by qRT-PCR;(6)Take two groups of cells(MRL / lpr mouse BMMSCs,shRNA lentivirus infected BMMSCs)to induce osteoblasts for 2 weeks and induce them into osteoblasts(OB),and use qRT-PCR to detect the difference of osteogenic indexes before and after infection(7)Take 10 MRL / lpr mice,the experiment is divided into 2 groups,5 MRL / lpr mice in each group,which are tail vein injection PBS lupus mice group,tail vein injection N-Cadherin shRNA lentivirus infection BMMSCs The lupus mice group were sacrificed at 20 weeks of age.Peripheral blood was taken from the eyeballs,and the blood routine differences of the mice in each group were detected by blood cell detector.Flow cytometry was used to analyze the ratio of bone marrow and spleen cells,and the colony formation test was used to detect the difference in the number of bone marrow and spleen colonies.Result:(1)The expression level of N-cadherin mRNA on the surface of MRL / lpr lupus BMMSCs was significantly higher than that of control group C57 BL / 6 mice(P <0.05,n = 5);(2)The expression of N-cadherin mRNA in MRL / lpr lupus BMMSCs 2 weeks after osteogenic induction was higher than that in the control group(p <0.001,n = 5);(3)The expression levels of COL1(P <0.05,n = 5)and RUNX2(P <0.001,n = 5)mRNA of MRL / lpr lupus BMMSCs were lower than those of C57 BL / 6 mice;(4)The mRNA expression of OB(COL1,ALP,OCN)in lupus mice is lower than that of C57 BL / 6 mice(P <0.05),and the mRNA expression of RUNX2(P <0.01)is lower than that of C57 BL / 6 mice,(5)The results of qRT-PCR showed that the expression of N-Cadherin in the shRNAN group was significantly decreased compared with the normal group of BMMSCs and the NC group(p <0.001,n = 5);(6)Osteoinduction of two groups of cells(MRL / lpr mouse BMMSCs,shRNA lentivirus infected BMMSCs)was induced to OB,and qRT-PCR test results showed that the expression of RUNX2 mRNA in lupus mouse OB was significantly increased compared with before infection(P <0.001,n = 5),the mRNA expression of COL1,ALP,OCN has an increasing trend compared with before infection,and has no statistical significance;(7)The results of tail vein injection transplantation showed that the WBC,RBC,HB,and PTL counts in the BMMSCs group increased compared with the PBS group,but there was no statistical significance;the flow cytometry results of the bone marrow and spleen showed that the BBMMSCs group B220 + B lymphocytes(P <0.05,n = 5),CD138 + plasma cell ratio(P <0.05,n = 5)decreased,the difference was statistically significant,compared with PBS group,BMMSCs group Ter-119 + erythroid,CD3 e + There was no obvious abnormality in the proportion of T lymphocytes and CD11 b myeloid system;bone marrow colony results showed that the CFU-pre-B(P <0.05,n = 5)decreased compared with PBS group and BMMSCs group,and the difference was statistically significant;compared with PBS group,There were no obvious abnormalities in BFU-E and CFU-GM in the BMMSCs group;spleen colony results showed that: compared with the PBS group,there were no obvious abnormalities in the BFU-E and CFU-GM in the BMMSCs group.Conclusion:(1)The expression of N-Cadherin on the surface of BMMSCs in MRL / lpr lupus mice is significantly higher than that in C57 BL / 6 mice;the expression of N-Cadherin in MRL / lpr lupus mice OB is higher than that in the control group;(2)The bone formation ability of MRL / lpr lupus mice is lower than that of C57 BL / 6 mice;(3)After down-regulation of N-Cadherin,the expression of N-Cadherin in MRL / lpr lupus mouse OB decreased significantly compared with that before down-regulation,and the osteogenic capacity of MRL / lpr lupus rat OB increased;(4)Down-regulation of BMMSCs N-Cadherin can inhibit the production of bone marrow,splenic B lymphocytes and plasma cells in MRL / lpr lupus mice and improve anemia.