The Studys Of TLR4 And CD14 In Sjogren's Syndrome Pathogenesy

PartⅠThe blood serum level of LPS,IL-6,TNF-αin patient with Sjogren's syndrome and the relative studyObjective:to observe the blood serum level of LPS,IL-6,TNF-αin patient with Sjogren's syndrome(SS) and the relative study.Methods:according to patient's condition,31 patient with Sjogren's syndrome were divided into three groups:initial group,relieve group and recur group; according to ESR and CRP,initial group was divided into two groups:acute inflammation group and non-acute inflammation group.used tachypleus amebocyte lysate to detect the level of lipopolysaccharide(LPS);IL-6,TNF-αwere detect by enzyme-labeled immunosorbent assay(ELISA),and analysis their dependability.Result:①the blood serum level of IL-6,TNF-αin patient with Sjogren's syndrome were significantly higher than control(P＜0.01);the blood serum level of IL-6,TNF-αin initial group were significantly higher than that in relieve group (P＜0.01);the blood serum level of IL-6,TNF-αshowed no statisticaldifferent between initial group and recur group(P＞0.05).the level of LPS in recur group was significantly higher than that in initial group and relieve group(P＜0.01);the difference of others groups have no statistical different(P＞0.05);②linear regression analysis between the level of LPS and IL-6,TNF-α,ESR showed positive correlation(P＜0.05);③the blood serum level of IL-6 and TNF-αin acute inflammation group and non-acute inflammation group were significantly higher than control(P＜0.01);the level of LPS,IL-6 and TNF-αin acute inflammation group were significantly higher than that in non-acute inflammation group(P＜0.01);but the level of LPS in non-acute inflammation group showed no statistical different between control(P＞0.05).Conclusion:①LPS have participated SS pathogenesy,infection is one of reasons for SS onset and recur;②IL-6 and TNF-αparticipated SS pathogenesy,the level of IL-6 and TNF-αchanged along with SS pathogenetic condition,so they can be indexes for SS pathogenetic condition.PartⅡThe expression of TLR4 and CD14 in peripheral blood monouclear cells of patient with Sjogren's syndrome Objective:to study The expression of toll like receptor 4(TLR4) and CD14 in peripheral blood monouclear cells(PBMC)of patient with Sjogren's syndrome.Methods:the expression of TLR4 and CD14mRNA was detected by reverse transcription reaction(RT- PCR);the expression of TLR4 and CD14 protein was detected by flow cytometry.Result:The expression of TLR4 and CD14 mRNA and protein in patient with SS were significantly higher than control(P＜0.05); The expression of TLR4 and CD14 mRNA and protein in initial group and recur group were significantly higher than relieve group(P＜0.05);the expression of TLR4 and CD14 mRNA and protein in initial group and recur group have no statistical different(P＞0.05).Conclusion:①the PBMC of patient with SS highly expressed TLR4 and CD14;the expression of TLR4 and CD14 coincidenced with SS pathogenetic condition.②LPS-TLR4 signal pathway have participated SS pathogenesy.PartⅢThe expression of TLR4 and CD14 in salivary gland of patient with Sjogren's syndromeObjective:to study the relation between TLR4,CD14 and salivary gland lesion.Methods:The expression of TLR4 and CD14 in salivary gland were detected by immunohistochemical method,the expression of TLR4 and CD14mRNA was detected by RT- PCR.Result:①the salivary gland of SS highly expressed TLR4 and CD14,the control group have no expression of TLR4 and CD14;②in 0～1 patho-grade:the expression's positive rate of TLR4 and CD14 is 62.5%;in 2 patho-grade:that is 70%;in 3 patho-grade:that is 75%;in 4 patho-grade:the salivary gland lesion is severe,the expression's positive rate of TLR4 and CD14 is decreased to 58.3%.the expression's positive rate of TLR4 and CD14 between every two groups have statistical different.③in PSS group,64.4%salivary gland ductal epithelial cells expressed TLR4 and CD14,67.8%lymphocyte in lymphocyte focus expressed TLR4 and CD14,the expression of TLR4 and CD14 in salivary gland ductal epithelial cells and lymphocyte focus showed no statistic different(P＞0.05);④the expression of TLR4 and CD14 mRNA in SS group were significantly higher than control(P＜0.05);Conclusion:①the salivary gland of patient with SS highly expressed TLR4 and CD14;the signal pathway of TLR4,CD14 maybe one of reasons for salivary gland lesion;②salivary gland ductal epithelial cells are active participants in SS pathogenesy,and they aren't passive sufferers;salivary gland ductal epithelial cells have the same biological effect with infiltrative lymphocyte in salivary gland. PartⅣThe effect of TLR4,CD14 signalpathway in salivary gland epithelial cellsObjective:to study the effect of TLR4,CD14 signalpathway in salivary gland epithelial cells. Methods:after culture salivary gland epithelial cells in vitro one week,①the PSS group cells and control all treatmented with1μg/ml LPS,at 1 h,8 h,12 h,24 h,48h,72 h,we extract cell's RNAand detected TLR4 and CD14mRNA by RT- PCR to study the LPS effect to salivary gland epithelial cells in different time;②the PSS group cells treatmented with different final concentration LPS:1ng/ml,10ng/ml,10~2ng/ml,10~3ng/ml,10~4ng/ml,10~5ng/ml,detected TLR4 and CD14mRNA by RT- PCR after 24h to study the LPS effect to salivary gland epithelial cells in different concentration;③using the best LPS final concentration-----1μg/mland the best culture time-----8h continue the experiment,we elected the best grow salivary gland epithelial cells and divided them into three group:1) LPS group:treatmented the cell with 1μg/ml LPS;2) interrupted group:,befor treatmented with 1μg/ml LPS,the cells were firstly treatmented with 10μg/ml HTA125 for 1h;3) control:no treatment.after culture 8h,we detected NF-κBp65 mRNA by RT- PCR;the expression of NF-κBp65 protein detected by Western Blot and TNF-α,IL-6 detected by ELISA.Result:①we certified the cultured cells in vitro source from salivary gland epithelial cells by cytokeratin staining;②the expression of TLR4 and CD14 mRNA were strengthen after treatmented with LPS for 1h,the peak time in 8h,after 24h the expression of TLR4 and CD14 fall-off;③the expression of TLR4 and CD14 mRNA were strengthen treatmented with 1ng/ml LPS,then the expression gradually strengthen with the heightened LPS concentration,10ng/ml LPS made the expression reached to the peak;④the expression of NF-κBp65 mRNA and protein in interrupted groupwere minimum,and that is maximum in LPS group(P＜0.01)⑤the level of TNF-αand IL-6 in interrupted group were minimum,and that is maxlmum in LPS group(P＜0.01).Conclusion:①salivary gland epithelial cells high expressed TLR4 and CD14mRNA,after treatmented with LPS,the expression were strengthen;②the signal pathway:LPS- TLR4- CD14-NF-κB-mediators of inflammation have participated PSS pathogenesy and further supports the role of infection in pathogenesis of PSS.