The Research And Its Significance Of Glycosylation Pattern Of Serous Glycoprotein In Systemic Lupus Erythematosus

CHAPTER ⅠMDR1 rs1128503 and rs 1045642 polymorphisms in systemic lupus erythematosus patients of Guangxi populationObjective To study the single nucleotide polymorphisms(SNP) of MDR1 gene at position rs1128503 (C1236T) and rs1045642 (C3435T) in healthy population and systemic lupus erythematosus(SLE) patients. To investigate whether rs1128503 and rs1045642 polymorphisms are associated with susceptibility to SLE of Guangxi population.Methods The polymorphisms of rs1128503 and rs1045642 were analyzed using the polymerase chain reaction-restriction fragment length polymorphism method(PCR-RFLP) in 283 patients with SLE and in 251 healthy controls. Different SNP genotypes and allele frequency distribution were used for testing of associations between polymorphisms and susceptibility to SLE.Results The genotype distribution of polymorphism of rs1128503 and rs1045642 in both groups was consistent with the Hardy-Weinberg equilibrium, that means the samples were representative and comparable(P>0.05). For rs1128503, statistical analysis showed no differences in genotypic frequencies distribution between SLE and healthy subjects (P>0.05). Nevertheless, the T allelic frequency was higher in SLE patients compared with healthy controls (P<0.05). Moreover, SLE patients had significantly increased chance carrying T allele (T vs. C, OR= 1.36,95%CI= 1.07-1.74, P= 0.014) and TT genotype (TT vs. CC, OR= 1.77,95%CI= 1.08-2.88, P= 0.022) compared with healthy population, especially for patients above 40 years of age.Conclusions These data suggest that susceptibility to SLE of Guangxi population may be influenced by MDRl rsl 128503 polymorphism. T allele and TT genotype of MDRl rs1128503 may be danger factors for development of SLE. Larger multicenter studies and studies across other ethnic groups are needed to elucidate the contradictory implications of MDR1 polymorphisms with susceptibility to SLE. CHAPTER IICHAPTER ⅡAnalysis of glycosylation pattern of serous glycoprotein from different stages of systemic lupus erythematosusBackground and Objective Systemic lupus erythematosus is a chronic autoimmune disease that is closely concerned with immune function. The abnormal immune response involved in the pathogenesis, progression and therapeutic effect of systemic lupus erythematosus. Glycosylation is a common post-translational modification, which play an important role in cell adhesion, signal transduction, structural stability, receptor construction, cell differentiation and immune regulation. In this part of study, we used lectin microarray to detect differential glycan profiling of serous glycoprotein from different active stages of systemic lupus erythematosus patients. Here, we aim to screen specific biomarkers that related to diagnosis or activity of systemic lupus erythematosus.Methods Patients were divided into 4 groups, including health group, untreated group, treated group, remission group. Experimental process shown in Figure 1, mainly includes the following steps:Equal volume sample were mixed to acquire pooled serum. Low-abundance protein was collected using high-abundance protein removal kits. After desalting, the low-abundance proteins were diluted with labeling buffer. The target proteins were labeled with biotin using Lightning-Link kits. After hybridized with labeled samples, lectin microarray containing 50 kind’s lectins were treated with fluorescence dye Cy5. Lectin microarray chips were scanned to collect affinity signal. Then, experimental data were analyzed with one-way ANOVA (Student-Newman-Keuls test) or non-parametric statistics (Rank-sum test) according to the homogeneity of variance.Results In this study, three independent chip data were analyzed. Intra-assay coefficient of variation<15%, inter-assay coefficient of variation <25%. In both 4 groups, statistically significant differences were shown in 20 kinds of lectins, including AAL, HAL, PSA, RCA60, SNA, EEL, LEL, NML, VAL, CFL, ConA, JAC, MPL, PCL, PHA-E, MAL-I, HHL, LCA, RCA-I, STL. Compared with health group, untreated group showed significantly reduced affinity in AAL, HAL, PSA, RCA60 and SNA (P<0.05), that suggested the decrease of terminal fucose, Sialylated Lewis antigen, N-acetylgalactosamine, a-mannose and a2,6 sialic acid of serous glycoprotein. Meanwhile treated group and remission group showed increased affinity in the 5 above lectins compared with untreated group (P<0.05), suggesting corresponding glycan of these lectins resumed. Untreated group also showed increased affinity in EEL, LEL and NML (P<0.05), which meant the increase of al,3 galactose, N-acetylglucosamine and high mannose. Treated group and remission group showed decreased affinity in these 3 lectins compared with untreated group (P<0.05). To VAL, CFL and MPL, there have not significant difference of affinity between untreated group and health group (P>0.05), while affinity signals increased greatly in treated group and remission group (P<0.05). Affinity signals of ConA, JAC, PCL and PHA-E increased significantly in untreated group compared with health group, and continued increasing in treated and health group(P<0.05). The rest 5 lectins, STL, MAL-I, HHL, LCA and RCA-I, also showed specific changes of affinity signals in different groups.Conclusions In this part of study, a variety of lectin affinity occurred in different stages of systemic lupus erythematosus, suggesting that specific glycan patterns of serous glycoprotein might be efficient biomarkers of diagnosis or progression of SLE. Terminal fucose, Sialylated Lewis antigen,N-acetylgalactosamine, a-mannose and a2,6 sialic acid decreased in untreated SLE patients compared to healthy people, while a 1,3 galactose, N-acetylglucosamine and high mannose increased greatly. Then after treatment, the synthesis of these sugar chains nearly resumed to normal levels, which supposed that these sugar chains might be related to the pathogenesis or progression of systemic lupus erythematosus.CHAPTER IIIVerification of glycosylation profiling of serous glycoprotein from different stages of systemic lupus erythematosus with immuno-blottingBackground and Objective Lectins, which considered as glycan "decoder", are widely used in glycomics and glycoproteomics. However, compared to the antigen-antibody reaction, the less affinity and specificity of biding between lectins and sugar chains are still the "bottleneck" of related technologies. Thus, the outcomes of lectin microarray chip often need to be verified with other methods to improve the accuracy and repeatability of the experimental data. In this part of the study, we tested and verified the chip data with lectin immunoblot technique using the same pooled serum detected in part Ⅱ and another batch of serum collected in the same entry criteria in order to assure the reliability of our outcomes of the chips.Methods The same pooled serum detected in part Ⅱ and another batch of serum collected in the same entry criteria were analyzed. Preparation of pooled serum samples and enrichment of low-abundance proteins were performed according to the same steps in part Ⅱ. Concentration of low-abundance proteins was determined after the standby. After SDS-PAGE electrophoresis, unknown proteins were transferred to PVDF membranes. The PVDF membranes were detected with ECL (Enhanced chemiluminescent) after HRP labeling was performed. The membranes were scanned and analyzed with Quantity One software.Results Six lectins were selected to perform lectin immuno-blotting, including SNA, Con A, PHA-E, LCA, PSA and AAL, using different batch of samples. The PVDF membranes were analyzed with Quantity One software and the content of sugar chain was represented with the intensity of protein band. Similar trend were shown in lectin immuno-blotting consistent with data of lectin chip in both two batches of samples. Verification of PHA-E using new batch of pooled serum had some differences compared to chip data. Outcome of lectin-blotting showed a low intensity in remission group, which was different from chip data. In both two sets of immuno-blotting, band intensity was differed from the chip’s fluorescence intensity.Conclusions Similar trend were shown in lectin immuno-blotting consistent with data of lectin chip in both two batches of samples, which could mostly confirm the accuracy and repeatability of our outcomes in this study. Individual differences and inter-assay error might result in inconsistencies in lectin immuno-blotting of PHA-E and microarray chip. Specific remains to be further investigated. Band intensity were differed from the chips’fluorescence intensity in both two sets of samples, the reason might be caused by different lectin dilution in blotting.CHAPTER ⅣEfficacy of sialylated intravenous immunoglobulin in systemic lupus erythematosusObjective To evaluate the efficacy of sialylated intravenous immunoglobulin (IVIG) combined with glucocorticoid and immunosuppressant in systemic lupus erythematosus (SLE).Methods We performed a search of electronic databases including PubMed, Embase, The Cochrane Library, CNKI, CBM, Wanfang database and Weipu database for all available randomized controlled trials(RCTs). The meta-analysis were performed by using Stata 12.0 software.Results We identified 5 RCTs involving 237 participants. The results showed that sialylated intravenous immunoglobulin combined with glucocorticoid and immunosuppressant in SLE markedly improved clinical remission(OR= 2.99,95%CI= 1.53-5.85, P= 0.001) and reduced the incidence of nosocomial infection(OR= 0.26,95%CI= 0.10-0.66, P= 0.005). However, there were no differences in either the SLE disease activity index(WMD=-1.80,95%CI=-3.96-0.35, P= 0.101) or the proteinuria(WMD =-1.09,95%CI=-2.20-0.03, P= 0.056) between the study and control groups.Conclusions Sialylated IVIG combined with glucocorticoid and immunosuppressant in SLE showed good efficacy in treatment for SLE patients.