| Aims:The first part of the study aims to find the related risk factors of gallbladder cancer and develop a nomogram for early diagnosis of stage I-II GBC.The second part of the study aims to clarify effects of Helicobacter pylori infection and 17β-Estrogen on proliferation,apoptosis,migration and oxidative DNA damage of HIBEC cell line in vitro.The third part of the study aims to clarify effects of Helicobacter pylori infection and 17β-Estrogen on oxidative stress related Nrf2/ARE pathway and apoptosis pathway of human biliary epithelial cell line HIBEC in vitroMethods:In the first part of the study,the nomogram was developed using logistic regression analyses based on a retrospective cohort of patients with stage Ⅰ-Ⅱ GBC The accuracy of the nomogram was validated by discrimination,calibration and a prospective cohort.In the second part of the study,after HIBEC was co-cultured with 17β-Estrogen(10-9mol/L)and H.pylori(MOI=1:1)and continuously passaged until its 15th generation,the following aspects of investigations were carried on:(1)The proliferation ability of HIBEC was measured by CCK-8 assay,plate clone formation assay and expression levels of Ki-67 detected by immunocytochemistry;(2)The cell apoptosis was detected by Annexin V/PI;(3)The cell migration ability was examined by Transwell assay;(4)The production of ROS and 8-OHdG were detected by flow cytometry and Immunofluorescence staining combined with confocal laser scanning microscope observation,respectively.The results were used to evaluate the level of oxidative stress as well as the related DNA damage in HIBEC cell.In the third part of the study,the following aspects of investigation were carried on:(1)The transcription and translation levels of Nrf2,Keap-1,NQO-1 and HO-1 were respectively measured by PCR and Western-blot;(2)The expression levels of Nrf2 in the nucleus of HIBEC were detected by double immunofluorescence labeling method as well as Western-blot in order to confirm its transposition into the nucleus;(3)The interactions between Nrf2 and Keap-1 was examined by the Co-IP assay;(4)Whether Nrf2 could bind to the DNA of NQO-1 gene was confirmed by EMSA assay;(5)The expression levels of proteins and enzymes essential for apoptosis pathway were detected by Western-blot.Results:Factors included in the nomogram were advanced age,hazardous alcohol consumption,long-standing diagnosed gallstones,atrophic gallbladder,gallbladder wall calcification,intraluminal polypoid lesion,higher wall thickness ratio and mucosal line disruption.The nomogram had concordance indices of 0.889 and 0.856 for the two cohorts,respectively.Internal and external calibration curves fitted well.Data of the second and third part of the study showed that 17β-Estrogen and H.pylori could either separately or together induce higher OD450 values,higher Ki-67 expression,higher colony forming efficiency(p<0.05);inhibit cell apoptosis(p=0.011 and 0.005,respectively);increase the number of cells permeating the septum(p=0.010 and 0.000,respectively)and the production of ROS and 8-OHdG(p=0.000).They could also significantly increase the transcription and translation levels of Nrf2,Keap-1,NQO-1,HO-1(p<0.05)and the expression levels of Nrf2 in the nucleus of HIBEC(p<0.01);Then,17β-Estrogen and H.pylori could induce the dissociation of Nrf2/Keap-1 complex(p=0.000),facilitate Nrf2 bind to the DNA of NQO-1 gene(p<0.05)and significantly suppressed expression levels of various cell apoptosis pathway related proteases(p=0.000).Conclusions:The proposed nomogram,based on the potential risk factors of gallbladder cancer,can provide valuable information for early diagnosis of stage Ⅰ-ⅡGBC.H.pylori and 17β-Estrogen,separately or in combination,could induce oxidative stress and related DNA damage,enhanced cell proliferation as well as suppression of apoptosis of HIBEC in vitro. |
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