| Part 1 The prevalence of PBC in 33,115 healthy persons Objective: To study the prevalence with primary biliary cirrhosis(PBC) in healthy persons.Methods: Serum type M2 anti-mitochondrial antibodies(AMA-M2) and liver function of 33,115 healthy persons were detected, and the prevalence of PBC, illness characteristics,age distribution and risk factors were analyzed.Results: 349 cases were screened as AMA-M2 positive, and 36 of them were diagnosed as PBC. The overall prevalence was 108.71/105. For female, the prevalence was 166.66/105(27 cases), higher than that of male which is 53.21/105(9 cases)(P<0.01). 36 PBC onset patients had an average age of(57.00±14.05). The prevalence of PBC was 18.79/105 ?131.00/105 and 440.14/105 in ?40?41~60 and?61 years of age groups, respectively.Prevalence difference were significant among three age groups(P<0.01). The prevalence of ?40 age group is lowest while that of ?61 age group is highest in all groups(P<0.01).The level of alkaline phosphatase(ALP) in PBC patients were significantly increased(223.01±160.67) U/L, among which 9 cases with ALT elevated from mildly to moderately,4 cases with ALT rose above 100 U/L and 12 cases with increased direct bilirubin(DBil).There was no imaging performance of intrahepatic bile duct obstruction. Moreover, 313 cases only showed the positive AMA-M2 but the ALP remained normal as well as no PBC related clinical symptom was found.Conclusion: Prevalence rate of PBC was not as rare as expected before in physical examination population. The key of screened PBC was used a method with high sensitivity.Part of only AMA-M2 positive cases may be patients in early stage of PBC. Follow-up should be carried out and disease progression should be paid close attention.Part 2 Preliminary Study on NF-?B signaling pathway associated with PBC pathogenesisObjective: To detect the proliferation of CD4+T cells stimulated by IL-18 and the activation of NF-?B signaling pathway, and explore the mechanisms of NF-?B signaling pathway in the PBC.Methods: Peripheral blood of 30 healthy controls, 30 only AMA-M2 positive cases and31 PBC patients were collected, then CD4+T cell were sorted with magnetic bead separation. Cell purity and number were detected by flow cytometry and cell count. The standard curve was made to select the best condition for cell culture, then the effect of IL-18 on the proliferation rate of CD4+T cells was detected by CCK-8 test. After IL-18 stimulation, the CD4+T cells were collected and the cytoplasm protein and nuclear proteins were extracted. Western Blot was used to detect the protein expression of I?B? and NF-?B p65 before and after the stimulation. After that, reducing rate of I?B? and increasing rate of NF-?B p65 were compared to understand NF-?B signaling pathway activation status in each study group. The abnormal characteristics of PBC patients' liver function were analyzed.Results: 1.With flow cytometry, CD4+T cell sorting purity is above 90%, and the separation efficiency was completely and high. 2. CD4+T cell count and CCK-8 cell proliferation assay: CD4+T cells of PBC group(0.286±0.051)×106/ml were significantly more than the other two groups(0.190±0.034)×106/ml,(0.182±0.042) ×106/ml(P<0.01).105 cells/well-inoculation and 4h-incubation with CCK-8 reagent was the optimum condition for CD4+T cell proliferation experiment. The result showed that CD4+T cells can be stimulated by IL-18 to proliferate, and the proliferation rate of PBC group(23.85%) was higher than the other two groups(11.75%, 12.98%)(P<0.01). 3. Western blot : Before IL-18 stimulation, the I?B? protein expression was the lowest(P<0.01) and the NF-?B p65 protein expression was the highest in PBC group than the other two groups(P<0.01). After IL-18 stimulation, I?B? protein reducing rate were 15.25%(control group), 13.37%(only AMA-M2 positive group), 34.37%(PBC group), respectively. Between the control group and the only AMA-M2 positive group, there was no significant difference of I?B? protein reducing rate(P>0.05), and the reducing rate of PBC group was higher than the other two groups(P<0.01). However, after IL-18 stimulation, NF-?B p65 protein increasing ratewere 23.91%(control group), 20.77%(only AMA-M2 positive group), and 61.35%(PBC group). Between the control group and the only AMA-M2 positive group, there was no significant difference of the NF-?B p65 protein increasing rate(P>0.05), and the increasing rate of PBC group was higher than the other two groups(P<0.01). 4. Liver function results of PBC group were analyzed: Main abnormal liver function performance was the significant increase of ALP, ?-GT, TBil and DBil as well as the mild or moderate increase of transaminase.Conclusion: 1. For PBC cases, there were abundant CD4+T cells in peripheral blood which have high proliferation ability and high sensitivity to IL-18 stimulation; 2. IL-18 could stimulate the proliferation of CD4+T cells, which may play an important role in the pathogenesis of PBC. 3. NF-?B signaling pathway was activated in PBC patient, and the activation of NF-?B signaling pathway was significantly promoted after IL-18 stimulation,which may be one of the mechanisms involved in the pathogenesis of PBC. |
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