| [Objective]To investigate molecular mechanism of benign bile duct scar, to provide clue of pathogenesis in bile duct hyperplastic scar, to confer the lab evidence of clinical research and therapy bile duct hyperplastic scar.[Methods]1. Normal fibroblast and Scar fibroblast were cultured by primary culture. MTT test was utilized to test the vitality of the cells.2. Test the normal fibroblast and scar fibroblast of differences proteins by microarray, differences proteins were handled by pathway-enrichment analysis and function-enrichment analysis.3. Differences proteins(Act B、TGF-β1、ET-1、Tsp-1 and OSM) were tested by ELISA test.4. siRNA-Act B was transfected to SCFB. The cell apoptosis was determined by flow cytometry instrument. The expression of Act B, Smad2/3, TGF-β1, ET-1, Tsp-1 and OSM were detected by Western blot.5. NFB was incubated with rh-Act B. The cell cycle was determined by flow cytometry instrument. The expression of Act B, Smad2/3, TGF-β1, ET-1, Tsp-1 and OSM were detected by Western blot.6. The cell was incubated with 12 kinds of bile acids. The vitality was determined by MTT method.7. The cell was incubated by CA or DCA, the cell cycle was determined by flow cytometry instrument.8. The count, vitality and distance healing of the scar fibroblasts with the CA plus DCA were tested.9. The cell was incubated with UDCA, the cell apoptosis was determined by flow cytometry instrument.10. The cell was incubated with UDCA, the expression of Act B, Smad2/3, TGF-β1, ET-1, Tsp-1 and OSM were detected by Western blot.11. Established of rabbit model for benign stricture of the common bile duct by absorbable surgical suture transfixion-ligation. 12. Liver function was tested.13. Scar elevation index (SEI) and area density on area (AA) were tested by Hematoxylin-Eosin stain and Masson trichromic stain.14. The expressions of Activin B and Smad2/3 were measured by the immunohistochemistry. 15. The expression of Scar tissue of Act B, Smad2/3, TGF-β1, ET-1, Tsp-1 and OSM were detected by Western blot.[Results]1. Succeed in culture the normal fibroblast and scar fibroblast of human benign biliary duct cicatrix. The vitality of scar fibroblast was higher than normal fibroblast. 2. Microarray data were collected from normal fibroblast and scar fibroblast.37 kinds of differences proteins were found by microarray (P<0.05).27 proteins were up-regulated and 10 proteins were down-regulated in scar fibroblast (P<0.05). Their function were associated with signaling by Activin, synthesis and degradation of extracellular matrix, formation and activation of cytokine, inflammatory reaction, immunoreaction, tissue damage reaction, cell cycle, migration, apoptosis and secretion, etc.3. The results of ELISA are same as microarray (P<0.05).4. The expression of Act B mRNA level was decreased by siRNA-Act B (P<0.05). The percent of early apoptosis was increased by siRNA-Act B. (P<0.05). The expression of Act B, Smad2/3, TGF-β1 were decreased and Tsp-1, OSM were increased by siRNA-Act B (P<0.05).5. The percentage of G0/G1 stage was decreased and S stage was increased by rh-Act B (P>0.05). The expression of Act B, Smad2/3, TGF-β1 were increased (P<0.05) and Tsp-1, OSM were decreased (P<0.01) by rh-Act B.6. Low concentration CA and DC A promote the vitality of SCFB (P<0.05).7. The percentage of G0/G1 stage was decreased and S stage was increased by CA or DCA (P<0.05).8. The count and vitality of the scar fibroblasts were significantly higher than the fibroblasts without CA or DCA (P<0.05).9. UDCA promotes the early apoptosis of SCFB (P<0.05).10. The expression of Act B, Smad2/3, TGF-β1 were decreased and Tsp-1, OSM in SCFB were increased by UDCA (P<0.05).11. Established of rabbit model for benign stricture of the common bile duct by absorbable surgical suture transfixion-ligation.12. Liver function was gently improved by UDCA (P<0.05).13. SEI and AA were gently decreased by UDCA (P<0.05).14. With the extension of the time, the expression of Act B and Samd2/3 were decreased by UDCA (P<0.05).15. With the increase of time, the expression of Act B, Smad2/3, TGF-β1, ET-1 were decreased and Tsp-1 was increased by UDCA (P<0.05).[Conclusions]1. There have differentially expressed proteins between SCFB and NFB. Activin B、TGF-β1、ET-1、Tsp-1 and OSM have refer to bile duct hyperplastic scar.2. Activin B signal plays an important role in the process of NFB transforming to SCFB. TGF-β1、Smad2/3, Tsp-1 and OSM are important participant.3. Low concentration CA and DCA promote the vitality of SCFB.4. UDCA promotes the apoptosis of SCFB. The mechanism may be that Activin B regulated the expression of TGF-β1, Smad2/3, Tsp-1 and OSM.5. The model of Liver function, SEI and AA were gently improved by UDCA, and the effects to have relations with time. The mechanism is that Activin B regulated the expression of TGF-β1, Smad2/3, ET-1 and Tsp-1. |
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