| The first part Expression fetures of Emp in different organs of lupus miceObjective Observed Erythroblast Macrophage Protein(EMP)expression in different tissue in lupus mice group and control mice groupMethod : 10 female MRL / lpr mice,aged 12 weeks,weighing 25.1 to 30.4(average 27.6)g,were purchased from Shanghai slac laboratory animal ceter.C57BL/6 mice aged 12 weeks,weighing 25.1 to 30.4(average 27.6)g,were purchased from Nanjing University animal experiment center.The animal raised in Guangdong experimental animal center,killed after 1 weeks adaption.The limbs muscles of the mice were stripped.Take the humerus and the femur,and the humerus long bones were placed in the decalcified fluid for 72 hours,then fixed to paraffin for immunohistochemical examination.The femur cartilage were stripped from both ends to expose the red marrow cavity.Operate on ice,1ml syringe was used to absorb PBS solution and rinse the marrow cavity for 2-3 times.1000r/min and 5min were centrifugally removed to the supernatant,and the total RNA was extracted for EMP gene expression detection.Then we took the liver,kidney,lung,spleen and brain tissue of mice,and put one part into formaldehyde to make EMP immunohistochemical(IHC)and double immunofluorescent(IF).The other part was placed in liquid nitrogen for EMP gene expression detection.ResultsQ-PCR detection revealed that EMP expression in the liver(t=2.747,p=0.017)and bone marrow(t=3.853,P=0.008)of lupus mice was significantly higher than in the control mice.However,no differences in EMP expression were observed in the renal,spleen,lung,and brain tissues between the lupus and control mice(P > 0.05).Additionally,EMP was expressed in all of the tissues of lupus mice,With the highest levels observed in the bone marrow.But there was no statistical difference between these tissues.The IHC results showed that EMP is ubiquitously expressed in all of the tissues of the lupus and control mice.The positive expression rate in the bone marrow and liver tissues of the lupus mice was higher than in the control mice,but without an obvious difference in the other tissues.The double IF staining method shows that EMP was expressed in macrophages in the tissues o f the lupus mice and the control miceConclusions EMP plays an important role in the pathogenesis of SLE.EMP is an important functional protein in the pathogenesis of SLE and the upstream target of possible targeted therapy.The widespread distribution of EMP in macrophages may be related to the extensive organ damage of systemic lupus erythematosus.The second part Relationship between EMP and antinuclear antibody formationObjective Silenced EMP expression by inject bone marrow macrophage carrying EMP-sh RNA adenovirus through Caudal vein,and observed ANAs,IL-1? in blood serum.To investigate the effect of bone marrow EMP on ANAs formation and immune inflammation.Method : 30 SPF grade MRL/lpr female lupus mice,aged 12 weeks,weighing 2 6.5~32.6(average 28.7)g,were purchased from Shanghai slac laboratory animal ceter.randomly divided into three groups: 1.Gene silencing group(injected bone marrow macrophage which injecting EMP-sh RN A adenovirus,n=10),2.empty carrier group,(injected bone marrow macrophage which injecting empty carrier adenovirus n=10),blank group(injected bone marrow macrophage,n=10).Three groups of mice were treated for 2 weeks.The remaining five lupus mice were used to extract mouse bone marrow macrophages.Observed and recorded the mice body weight,physical activities,change of medication during the treatment.After 2 weeks,take the mice femur,use Q-PCR to detect the expression of EMP in three groups.The serum of three groups of mice were taken to detect the co ncentration of anti nuclear antibody protein.Results The results of EMP expression in each group showed that the EMP expression of bone marrow macrophages in the gene silencing group was significantly lower than that in the blank group.The difference was statistically significant(T=4.852,P=0.01).The EMP expression of bone marrow macrophages in the gene silencing group was lower than that in the empty carrier group.The difference was statistically significant(P T=6.778,P=0.003).The expression of EMP in bone marrow macrophages in empty carrier group was not significantly different from that in blank group(P > 0.05).The ELISA detection shows that: concentration of serum ANA in interference group was significantly higher than empty adenovirus vector group,the difference was statistically significant(T=3.703,P=0.01);empty adenovirus vector group serum ANA concentrations higher than those in control group,the difference was statistically significant(T=3.902,P= 0.005).The concentration of serum AN A in the interference group was not significantly different from that in the blank group(P > 0.05).(P>0.05).The concentration of serum IL-1? in the interference group was significantly lower than that in the empty adenovirus vector group,and the difference was statistically significant(T=3.944,P=0.002).The concentration of serum IL-1? in the empty adenovirus vector group was higher than that in the control group,and the difference was statistically significant.(t=3.037,P=0.011).The concentration of serum IL-1 ? in the interference group was not significantly different from that in the blank group(P > 0.05).Conclusion The decrease of anti-nuclear antibody and IL-1 ? concentration in gene silencing lupus mice suggests that EMP is involved in the formation of ANA.It is not clear whether EMP affects the immune inflammation related factors through the formation of ANA.It is a great value for us to further confirm whether the formation mechanism of ANA is the core pathogenesis of SLE.It is worth our further study.The full text conclusion EMP widely expressed macrophages in various tissues and participated in the formation of AN A in the core pathogenesis of SLE.We believe that SLE may be an important upstream functional protein in the pathogenesis of SLE and an important upstream target antigen in the treatment of SLE,which is worthy of further study. |
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