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Objective To observe the effect of α‐Fodrin siRNA on human salivary gland(HSG)cells and to discuss its thera‐py on sj?gren′s syndrome(SS) .Methods The vectors expressing siRNA againstα‐Fodrin of human were transfected into HSG cells of 10μg α‐fodrin siRNA1 group andα‐Fodrin siRNA2 group ,while pGFP‐V‐RS vector were transfected into the cells of empty vec‐tor group ,there was no handling in HSG cell of control group .The efficiency was observed by fluorescence microscope after trans‐fection of 24 ,48 ,72 and 96 h by lipofectamine 2000 .The expression levels of α‐Fodrin mRNA and protein of HSG were detected by real‐time(RT)‐PCR and immunohistochemistry method respectively .The expression levels of IFN‐γ and IL‐10 in supernatant of cells were detected by ELISA .Results The efficiency was highest on 48 h after transfection .The level of α‐Fodrin mRNA and pro‐tein was lower in α‐fodrin siRNA1 group and α‐fodrin siRNA2 group than control group and empty vector group on 48 h after transfection (P0 .05) .The levels IFN‐γ in α‐fodrin siRNA1 group and α‐fodrin siRNA2 group were lower than of control group and empty vector group on 48 h after transfection ,but there were no significant differences in the four groups (P>0 .05) .Conclusion Theα‐fodrin siRNA1 andα‐fodrin siRNA2 can suppress the levels of α‐fodrin mRNA and protein of HSG cells ,at the same time;they can elevate the expression of IL‐10 and decrease the level of IFN‐γ.Soα‐Fodrin siRNA reduce the levels of inflammatory cyto‐kines and provide experimental basis to therapy of SS .
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Objective To construct two vectors of small interfering RNA (siRNA) expressing α-Fodrin and investigate its therapeutic effects on mice model with primary Sj(o)gren's syndrome (non-obese diabetic mice,NOD mice).Methods Sixteen 8-week-old NOD mice were randomly divided into four groups:the control group,the vector group,the α-Fodrin-siRNA1 group and α-Fodrin-siRNA2 group,4 mice in each group.Four template DNA of α-Fodrin siRNA were chemically synthesized and annealed to two double stranded (dsDNA),then digested by BamH Ⅰ and Hind Ⅲ.The digested double strands oligos were inserted into the downstream of U6 promoter of linearized pGFP-V-RS vector.Recombinant were confirmed by restrictive enzyme digestion and sequencing.Then the vectors were injected throughtail veil once a week,two times in total,while mice in the control group were injected with the same dose of phosphate buffer saline (PBS)and the vector group were injected with the same dose of vector vehicle.pGFP-V-RS was labeled by green fluorescent protein(GFP) and lacriminal glands underwent pathological examination.In addition,the expression of α-Fodrin mRNA in lung were detected by reverse transcription-polymerase chain reaction (RTPCR),and α-Fodrin protein in lacriminal glands and lung were detected by immuno-histochemistry.Serum interferon (IFN-γ),interleukin-17 (IL-17) concentrations in each group were detected by enzyme linked immunosorbent assay (ELISA) in order to observe changes in cytokine levels.At the same time,the pathological changes of the lacriminal glands and organs with hematoxylin-eosin (HE) staining were observed.The repeat ANOVA was used for statistical analysis.Results ① We constructed two siRNA eukaryotic expression vector successfully; ② α-Fodrin-siRNA could target to the lacriminal glands.③ Compared with the control group and vector vehicle group,the expression of α-Fodrin mRNA and protein were significantly decreased in the treatment groups.④ Compared with the control group [(11.73±2.73) pg/ml] and vector vehicle group [(15.40±1.99) pg/ml],serum IL-17 levels in the treatment groups were [α-Fodrin-siRNA 1 group (4.38±1.02) pg/ml; α-Fodrin-siRNA 2 group (4.55±0.06) pg/ml] significantly decreased (P＜0.05),but IFN-γ levels in the αt-Fodrin-siRNA group were not decreased significantly (P＞0.05).⑤ Compared with the control group and vector vehicle group,lymphocyte infiltration of lacriminal gland and inflammatory cell infiltration of alveolar and interstitial were significantly reduced in α-Fodrin-siRNA groups.Conclusion Specific α-Fodrin siRNA can inhibit the inflammation,and suppress the inflammatory infiltration of lacriminal glands and lung in mice with primary Sj(o)gren's syndrome.So the constructed vectors may slow the progression of pSS.
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Objective To evaluate the value of IgA and IgG antibodies against α-fodrin in both serum antibodies in SS is also assessed.Methods Samples from 39 patients with SS(25 primary and 14 secondary),8 patients with systemic lupus erythematosus(SLE),and 15 patients with rheumatoid arthritis (RA)as well as 10 healthy blood donors were collected.Anti-α-fodrin antibodies were measured using ELISA.Results The titer of serum anti-α-fodrin was higher in SS than in other connective tissue diseases group and healthy group(P<0.01).IgA type anti-α-fodrin antibodies was detected in 60%.44% of serum and saliva in patients with pSS respectively.IgG antibodies were detected in 43% of sera,and 29% of saliva of patients with pSS.The sensitivity and specificity of serum anti-α-fodrin IgA in SS was 54%and 85%.The level of anti-α-fodrin was positively associated with xerostomia and parotid swelling (P<0.05),and was negatively associated with xeroma,renal tubule acidosis,lung interstitial disease and hepatic damages(P>0.05).Conclusion Saliva and serLlm anti-α-fodrin level may be diagnostic for SS.It may be a useful screening marker.
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Objective To immunize BALB/c mice with type 3 musearinic acetylcholine receptor polypeptide (M3RP) and to evaluate the role of M3RP in SS. Methods Four-week-old BALB/e mice were immunized with M3R polypeptide 213-228 (M3RP) on days 0, 14, 35, 56, and were re-immuniged on days 65, 84, 105, and one mouse was killed every 2 to 3 weeks. The mice of the control group were immunized with submaxillary gland homogenate, GST, and PBS. The animals were analyzed for the presence of anti-SSA,anti-SSB, RF, ANA, anti-a-fodrin and anti-M3RP in sera by immunofluorescenee or ELISA. The cytokines of IFN-γ IL-2 and IL-10 were measured with ELISA. Salivary glands were examined by H＆E staining and immunohistochemical analysis. Volume of water drinking by each groups were calculated. Results BALB/e mice immunized with M3RP and submaxillary gland homogenate developed an immune response directed against M3RP, α-fodrin and ANA, but no antibodies against SSA, SSB and RF were found. Furthermore, lym-phocytic infiltrates in the salivary glands of immunized animals were observed 50 days after first immunization of M3RP and submaxillary gland homogenate. The serum IFN-α in mice of M3RP, submaxillary gland ho-mogenate, GST and PBS was (62±6), (89±5), (30±5) and (19±6) pg/ml respectively, and IL-2 was (12.6±1.6), (19.8±0.4), (3.9±0.9), and (4.9±1.1) pg/ml respectively (P<0.05). No difference was found in the level of serum IL-10 among the four group. Expression of α-fodrin was found only in submaxillary gland in M3RP and submaxillary gland homogenate groups of mice, but not in PBS and GST controls when studied by immunohistochemical analysis. Conclusion These results suggest that BALB/c mice immunized with M3RP are reminiscent of human SS, and M3RP as an autoantigen participates the development of SS.