| Background System lupus erythematosus(SLE) is a typical autoimmune disease, featured with a variety of autoantibodies production and multiple organ damage. The complicated clinical manifestations and prolonged course of SLE cause great impact on physical and mental health of patients, and has become a major public health problem. Previous studies have indicated that the occurrence of SLE was related to the high activation of T, B lymphocytes, increased immuneglobulin, and especially the proliferation, differentiation and functional abnormalities of B cell. B cells participate in many aspects of the pathogenesis of SLE by mediating tissue and organ damage through autoantibodies production, presenting antigen to autoreactive T cells and generating soluble mediators to trigger and maintain inflammations. NF-kB is an important transcription factor involved in intracellular signal transduction, which plays a key role on activation, proliferation of T cell and regulation of B cell immune globulin gene expression. Existing evidence shows that the activation of NF-kB signal pathway mediated by the B cell receptor(BCR) is involved in the pathogenesis of SLE. Protein kinase C beta(PKCβ) is a key factor in BCR mediated NF-kB signal pathway and plays an important role on the survival of mature B cell through the activation of this pathway. Bcl-x L and Bcl-2 are two members of the Bcl-2 protein family, which have the function of inhibiting cell apoptosis. Studies show that NF-kB can up-regulate the expression of anti-apoptotic proteins Bcl-x L and Bcl-2, and the shorter survival time of PKCβ defected B cell also related to the disorder expression of Bcl-x L and Bcl-2.Previous studies in the Central China and Southern China region have shown that the rs16972959 gene locus on PKC is related to the genetic susceptibility of SLE, but it is short of the report of the relationship between the PKCβ gene single nucleotide polymorphism of rs16972959 and SLE clinical features in Anhui Han population. Therefore, this study aims to detect the single nucleotide polymorphism of rs16972959 gene in SLE patients and healthy controls and to explore the relationship between PKCβ and genetic susceptibility of SLE in Anhui Han population. At the same time, using the ELISA method to detect the plasma levels of PKCβ, Bcl-x L and Bcl-2 in SLE patients, to investigate the relationship among them, and combined with clinical features, analyze the relationship between PKC, Bcl-x L and Bcl-2 plasma levels and SLE disease activity, between PKC, Bcl-x L and Bcl-2 plasma levels and different clinical and laboratory features, also analyze the relationship between PKCβ gene polymorphism and plasma expression level.Part Ⅰ Association Study of PKCβ Single Nucleotide Polymorphism with Systemic Lupus ErythematosusObjective To compare the distribution of genotype and allele frequencies of SNP rs16972959 of PKCβ gene between SLE patients and healthy controls in Anhui province and to explore the relationship between the distribution and the genetic susceptibility to SLE. At the same time, analyze the association between the distribution and the main clinical and laboratory features in SLE.Methods Totally, 400 patients with SLE were recruited. All of them are from department of Rheumatology and Immunology of the First Affiliated Hospitals of Anhui Medical University and Anhui Provincial Hospital. All patents fulfilled the 1997 revised criteria of the American College of Rheumatology for the classification of SLE. 400 cases of healthy blood donors were recruited as controls. Genotype and allele frequencies of SNP rs16972959 of PKCβ in SLE patients and healthy controls was detected by Taq Man SNP assay. And the differences of them between SLE patients and healthy controls were detected by Chi-square or Fisher’s exact test. SPSS 10.01 software was used to perform the statistical analyses. The test level α was 0.05.Results(1) HWE test: Genotype discrimination result of SLE and healthy controls were accorded with Hard-Weinberg equilibrium(χ2=1.523, P=0.217).(2) Genotyping of PKCβ(rs16972959): For SNP rs16972959, genotype frequencies of A/A, A/G and G/G in SLE patients were 3.75%, 33.75% and 62.50%, while 8.00%, 36.75% and 55.25% in healthy controls, respectively. And the difference of genotype frequencies between two groups were statistical significant(χ2=13.816,P<0.001). The frequency of minor allele A at SNP rs16972959 in SLE patients was lower than that in healthy controls, was 20.63% and 26.37%, respectively( χ2=7.356,P=0.007).(3) Association of PKCβ(rs16972959)polymorphism and clinical features No association were found between the distribution of A/A, A/G and G/G genotype of SNP rs16972959 and clinical features including butterfly erythema, discoid erythema, light sensitivity, oral ulcers, arthritis, arthritis, kidney disease, hematologic abnormalities(all P>0.05).(4) Association of PKCβ(rs16972959) polymorphism and laboratory features No association were found between the distribution of A/A, A/G and G/G genotype of SNP rs16972959 and laboratory features including anti-nuclear antibody(ANA), anti-double stranded DNA antibody, anti-Sm antibody, anti-SSA antibody, anti-SSB antibody, anti-ribonucleoprotein antibody(RNP)(all P > 0.05). But significant association was found between the distribution of A/A, A/G and G/G genotype of SNP rs16972959 and Anti ribosomal P protein antibody(χ2=6.361,P=0.042).Conclusion Our study suggests that PKCβ(rs16972959) gene polymorphisms is associated with the susceptibility to SLE, and G allele of SNP rs16972959 may increase the risk of SLE in the population studied.Part Ⅱ Association Study of PKCβ, BCL-x L and BCL-2 Plasma Levels with Systemic Lupus ErythematosusObjective To compare the difference of PKCβ, Bcl-x L and BCL-2 plasma levels between SLE patients and healthy controls, SLE nephritis group and non-nephritis group as well as between active and inactive SLE patients. To investigate the relationship between the plasma levels of PKCβ, Bcl-x L and BCL-2. At the same time, the relationship between the plasma levels of PKCβ, Bcl-x L and BCL-2 and clinical and laboratory features of SLE were also discussed.Methods A total of 80 patients with SLE from department of Rheumatology and Immunology of the First Affiliated Hospitals of Anhui Medical University were recruited, and collect their general information, clinical data and laboratory data. All patents fulfilled the 1997 revised criteria of the American College of Rheumatology for the classification of SLE. 80 cases of healthy blood donors were recruited as controls and collect their general information. Specific enzyme linked immunosorbent assay(ELISA) kits were used to detect the plasma level of PKCβ, Bcl-x L and BCL-2. The t-test of two independent samples was used to compare the differences of PKCβ,Bcl-x L and BCL-2 plasma levels in SLE group and the control group. All data were analyzed by SPSS 10.01 The test level α was 0.05.Results(1) No significant differences of PKCβ level in plasma between SLE patients and healthy controls(t=0.62,P=0.535). Bcl-x L plasma level in SLE patients were lower than healthy controls(t=6.00, P<0.001). Bcl-2 plasma level in SLE patients were higher than healthy controls(t=2.18, P=0.031)(2) There were no significant relationships between the plasma levels of PKCβ with the BCL-2 and Bcl-x L plasma levels in SLE patients and controls, respectively(all P>0.05); there were no significant relationships between the plasma levels of Bcl-x L and Bcl-x L plasma levels in SLE patients(rs=0.035, P =0.820); the plasma levels of Bcl-x L were inversely related to the Bcl-x L plasma levels in controls(rs=-0.342, P=0.007).(2) There were no significant differences of PKCβ, Bcl-x L and Bcl-2 plasma levels between the clinical features positive group and the negative group(all P>0.05).(3) There were no significant differences of PKCβ, Bcl-2 plasma levels between the laboratory features positive group and the negative group(all P>0.05). There were significant correlations between Bcl-x L plasma level with C4, Ig G, Ig M(P1=0.041, P2=0.024, P3=0.019) and no significant correlations with the other laboratory features(all P>0.05).Conclusion No significant difference of PKCβ level in plasma between SLE patients and healthy controls. Bcl-x L plasma level in SLE patients was lower than healthy controls. Bcl-2 plasma level in SLE patients was higher than healthy controls. |
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