| Systemic lupus erythematosus(SLE)is a kind of autoimmune diseases characterized by multiple organ damage.Lupus nephritis(LN)is a common complication of SLE,and it is also one of the main causes of disability.LN is an immune complex mediated nephritis,and its exact pathogenesis is not clear.Nowadays,it is believed that the pathogenesis of LN may be related to genetic factors and multiple immune cell dysfunction,such as T cells,B cells,monocytes and so on.In our previous genetic screening experiment,we found that TRIM38(tripartite motif-containing 38)m RNA expression levels was high in peripheral blood of untreated LN patients.We assumed that TRIM38 gene may be involved in the pathogenesis of LN,but there is no report about the expression of this gene in the recurrent LN,and no literature of its correlation with LN's disease activity and clinical parameters.Objective: Detected the expression of peripheral blood TRIM38 m RNA by quantitative real-time polymerase chain reaction(RT-PCR)in recurrent LN patients before and after treatment,observed the correlation TRIM38 m RNA with clinical indicators and explored its relationship with LN relapse.The aim of this study was to explore the possibility of TRIM38 as a molecular marker to predict the recurrence and prognosis of patients with LN.The detection of noninvasive markers may be of great significance for the prognosis and treatment of patients with recurrent LN.Methods: 1 Research subjects and grouping:From July 2015 to October 2016,25 patients with recurrent LN were recruited from the Department of Rheumatology and Immunology of the Second Hospital of Hebei Medical University.The clinical data of all 25 patients were used as the pre-treatment group.14 cases of recurrence of LN patients received two weeks after thecorticosteroids and immunosuppressive therapy as post-treatment group.All patients met SLE criteria of ACR(2009),and without hepatitis,tuberculosis and other infectious diseases,tumor and other connective tissue disease.All patients were completed the disease activity score(SLEDAI score)before and after treatment.32 cases with patients age and sexuality matched healthy volunteers were selected as healthy control group.All patients were approved by the ethics committee of the Second Hospital of Hebei Medical University and signed the written-informed consents.2 Research methods:Fasting blood samples were collected before and after treatment in the morning.The indexes of 24 hours urine protein,blood urea nitrogen(BUN),serum creatinine(Scr),immunoglobulin A(Ig A),immunoglobulin G(Ig G),immunoglobulin M(Ig M),complement 3,complement 4,triglyceride(TG),total cholesterol(TC),total cholesterol(TBIL)and other laboratory examination results were recorded separately.The total RNA was extracted from the peripheral blood and reversed transcription of c DNA.The target gene and reference gene(GAPDH)were amplified using q PCR technology.The results were recorded in a single PCR reaction to the logarithmic period amplification cycle number(cycle threshold,Ct).The ?Ct value was calculated as the target gene Ct value minus GAPDH Ct value,and the amount of gene expression is represented by 2-? Ct.And then we compared the difference of target gene expression level among healthy control group,pre-treatment group and post-treatment group.We also observed the correlation of TRIM38 m RNA and clinical indexes.3 Statistical analysis:All the data were analyzed by statistical software of SPSS 21.0.Measurement data of normal distribution was represented by(x ±s).If data did not coincidence with normal distribution,median(interquartile range)M(QR)was adopted.Compared two measurement data,and if data accorded with normal distribution and variance homogeneity,independent samples t test was adopted.If they did not coincidence with normal distribution,rank sum test(U)was used.Compared multiple independent samples and if they met with normal distribution,analysis ofvariance was used.If it did not satisfy with normal distribution,rank sum test(H)was selected.Pearson correlation analysis was used to analyze the correlation between two variables,and the Spearman correlation analysis was applied to the normal distribution.In order to study the influencing factors of TRIM38 gene expression,multiple linear regression analysis was adopted.Alpha=0.05 was taken for the test level.P < 0.05 was considered statistically significant.Results: 1 Total RNA of blood samples in three groups The RNA concentration of healthy controls was 49.5 ±50.28.That of LN patients was 84.60±74.06.The purity ratio of OD was about 2.08 ±0.04 on 260 nm / 280 nm.The results of 1% agarose gel electrophoresis showed that the 18 S and 28 s bands were clear,and the brightness of the 28 s was about 2 times of 18 s.This indicaded that the integrity of the RNA was good.2 TRIM38 m RNA amplification Agarose electrophoresis results showed TRIM38 m RNA stripe was a single and clear strip.It was consistent with the expected size of 172 bp.3 The results of TRIM38 m RNA after real-time fluorescent q PCR Real-time fluorescent quantitative RCR results showed that amplification was very well.The melting curve was in single-peak status and the peak value was 88.36.Electrophoresis results showed RNA stripe is only one single and the size is correct.4 The expression of TRIM38 m RNA in the three groups 4.1 No matter before treatment 0.768(0.690)and after treatment 0.587(0.472),the expression level of TRIM38 m RNA was higher than that in healthy control group 0.361(0.603).There was statistically significant compared with healthy control group(P < 0.05).4.2 Compared with the healthy control group,the expression of TRIM38 m RNA in patients with recurrent LN was significantly increased before treatment,and there was a significant difference between the two groups(P < 0.05).4.3 After treatment,the expression level of TRIM38 m RNA was lower than that before treatment,but there was no significant difference between the two groups(P > 0.05).4.4 After treatment,the expression level of TRIM38 m RNA was still higher than that of healthy control group,but there was no statistical significance(P > 0.05).5 Correlation between the expression level of TRIM38 m RNA and clinical indicators The expression level of TRIM38 m RNA was positively correlated with TG(r=0.500,P=0.015),and there was a significant negative correlation with complement C3(r=-0.513,P=0.009).But there were no correlation with complement C4,SLEDAI score,24 hours urine protein,BUN,Scr,Ig A,Ig G,Ig M,TC,TBIL(P > 0.05).6 Influence factors of gene expression level In multivariate linear regression analysis,we used stepwise regression analysis.The result showed that only C3 enter the equation,and the equation is: 2-? Ct=1.143-0.782 C3(P < 0.05).7 TRIM38 m RNA expression levels in patients with different anti ds-DNA antibody levels Patients were grouped by anti ds-DNA antibody level in the reciprocal ratio,and the expression levels of TRIM38 m RNA(0.887±0.714 vs 0.857±0.339)in patients with different levels of anti ds-DNA antibody were no significantly difference(P > 0.05).8 TRIM38 m RNA expression levels in different groups with 24 hours urine protein level The expression levels of TRIM38 m RNA were 0.763±0.463,0.577±0.302 and 0.832±0.4736 respectively in low-,mid-and high-urine protein group.There was no significant difference in different groups(P > 0.05).9 After treatment with glucocorticoid and immunosuppressive agents: SLEDAI score,BUN,Scr,TBIL were statistically significant(P < 0.05).There were no statistically significant for Ig A and 24 hours urinary protein,(P > 0.05).Conclusions: 1 The expression levels of TRIM38 m RNA in peripheral blood of patients with recurrent LN increased.It suggested that TRIM38 gene may be involved in the process of LN recurrence.This finding might have great significance to explore the pathogenesis of LN and maybe provide experience for the treatment of LN.2 The expression levels of TRIM38 m RNA decreased after treatment with glucocorticoid and immunosuppressive agents,but there was no statistical difference before or after treatment.Next step,we will expand the sample size in further research.3 TRIM38 gene probably involved in abnormal lipid metabolism of LN.4 TRIM38 may be unrelated to the severity of LN recurrence. |
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