| Part?In vitro study of fibroblast proliferation in simulant biliary environmentObjective To observe the proliferation of fibroblast which is one of the most important parts of bile duct repair by simulating the biliary environment as the intraluminal stent anastomosis of bile duct defect with autologous tissue in vitro.Methods Simulating the biliary environment by fibroblast cultured in vitro, and studying the function of bile during the bile duct repair procedure by observing the fibroblast proliferation through microscope, evaluating the proliferation by MTT method and analysising the apoptosis rate of fibroblast by double-labeled flow cytometry. The experimental group simulated the biliary environment with 1%bile and the control group added the same dose culture solution.Results Higher fibroblast proliferation was observed by microscope in experimental group over the control group after 48 hours being cultured in stimulant biliary environment. The OD570 value of fibroblast with different concentration was higher in experimental group than in control group after 48 and 72 hours being cultured in biliary environment showed a significant statistic difference (P<0.05). The apoptosis rate was lower in experimental group than in control group by double-labeled flow cytometry (P<0.05).Conclusion Bile can accelerate the fibroblast proliferation and the conversion to myofibroblast, and bile can inhibit the fibroblast apoptosis. It is considered that intraluminal stent end-to-end bile duct anastomosis can isolate the stimulus and influence of anastomoses from biliary environment, and to some extent decrease the bile duct excessive healing by fibroblast proliferation and convertion to myofibroblast because of the bile stimulation, which provide the theoretical basis for the next animal studies.Part?Animal studies of intraluminal stent repair of bile duct defect with autologous tissue in porcine modelObjective To assess the feasibility and safety of intraluminal stent repair of bile duct defect with autologous tissue.Methods A total of 30 Bama minipigs were randomly chosed and divided into two groups,15 in each. Experimental models with common bile duct (CBD) defect (1.0cm segment of CBD resected) were built by animal surgery, the experimental group was repaired by a kind of intraluminal stent with greater omentum, and the control group was formed to be repaired by end-to-end bile duct anastomosis. Incidence of jaundice and bile leakage was evaluated in both groups.Five pigs were killed each time at 2 weeks, 1 month, 3 months after the surgeries, CBC, CRP, Liver enzymes (ALT, AST, ALP) and serum total bilirubin were respectively compared preoperatively and at 2 weeks, 1 months, 3 months postoperatively in both groups.Pathomorphologic changes of the anastomoses were observed by HE & Masson staining, a-SMA, TGF-(31, b-FGF immunohistochemistry. Liver tissue slides with HE staining were analyzed. Cholangiography was performed for the randomly selected pigs at 1 month and 3 months postoperatively. The hydroxuproline of anastomoses was determined at 2 weeks, 1 month and 3 months postoperatively. a-SMA, TGF-?1, b-FGF mRNA expression of anastomoses were performed by RT-PCR at 2 weeks, 1 month, 3 months postoperatively.Results All operations were accomplished successfully. There was no postoperative jaundice and no bile leakage in both groups.Except the CBC results in control group at 2 weeks and 3 months postoperatively were higher than the experimental group (P<0.05), there was no significant difference comparing CBC and CRP tested preoperatively with those tested in the same animal at 2 weeks or 1 month or 3 months postoperatively in both groups (P>0.05)The ALT, AST, ALP results in control group at 3 months postoperatively were higher than the experimental group with significant difference (P<0.05). And there was significant difference comparing serum total bilirubin tested preoperatively with those tested in the same animal at 1 month or 3 months postoperatively in control group and at 3 months aftr the surgery in experimental group (P<0.05)Macroscopic appearance showed the stent in situ at 2 weeks and 1 month postoperatively in experimental group was with edema and congestion in anastomoses. 3 months after surgery the edema and congestion in anastomoses decreased with the stent degradated and disappeared. In control group, the anastomoses had mild edema and congestion at 2 weeks and 1 month postoperatively, and we also found mild expansion of the proximal bile duct and stricture of anastomoses 3 month after the surgery.The cholangiography showed no bile leakage or bile duct expansion at 1 month and 3 months postoperatively in experimental group. But in control group we observed some animals (1 in 5 at 1 month and 3months after surgery) have bile duct expansion without obstruction.HE staining showed anastomoses edema with inflammatory cells infiltration, most of which were macrophage and lymphocyte,and the collagen hyperplasia with small blood vessels proliferation at 2 weeks postoperatively. The level of collagen hyperplasia was higher in experimental group. At 1 month after the surgery both the edema and inflammatory cells infiltration decreased, and the collagen hyperplasia with fibrosis was observed which the structure of fibers was irregular. The neo-bile duct epithelial cells proliferation were with accessory glands. At 3 months postoperatively, microscopic appearance was like the 1 month postoperatively, but the fibers became less disorderly and the glands proliferation decreased. Less disorderly hyperplastic fibers and collagen prolification were observed in the experimental group while comparing the two groups at 1 month and 3 months postoperatively. No cholestasis and infiltrating inflammatory cells were found in the liver in both groups at 2 weeks, 1 month and 3 months postoperatively.Masson staining also showed the same results of collagen hyperplasia and fibers disorder as HE staining.The a-SMA positive expression scores showed significant difference at 1 month and 3 months postoperatively comparing with these two groups (P<0.05), scores had a trend of increase during the period. The TGF-?1 positive expression scores showed significant difference at 2 weeks after the surgery comparing with these two groups (P<0.05), scores had a trend of decrease during the period. The b-FGF positive expression scores showed significant difference at 1 month postoperatively comparing with these two groups (P<0.05), scores had a trend of increase first then decrease during the period and had a peak at 1 month postoperatively. A significant difference was found in the determination of hydroxyproline at 1 month and 3 months postoperatively while comparing the experimental group and the control group (P<0.05), but no significant difference at 2 weeks postoperatively (P>0.05). RT-PCR illustrated that,2 weeks after the operation, comparing to the control group, the expression of b-FGF, TGF-?1 mRNA in the experimental group increased, while the expression of a-SMA mRNA decreased; 1 month after the operation, the expression of b-FGF mRNA in the experimental group increased, while the expression of TGF-?1, a-SMA mRNA decreased; 3 months after the operation, the expression of b-FGF, TGF-?1 mRNA in the experimental group were corresponding roughly to the control group, but the a-SMA mRNA in the experimental group were less.Conclusion Intraluminal stent repair of bile duct defect with greater omentum as autologous tissue is a convenient, safe and feasible approach to reconstruct the bile duct defect. Animal experiment demonstrate that, comparing to the end-to-end anastomosis repair of bile duct defect, the intraluminal stent repair of bile duct defect with greater omentum as autologous tissue has its superiority in preventing bile duct excessive healing. But the degradation duration of the stent may be prolonged, and this approach still needs to be confirmed through long-term animal experiment with large samples and clinical research. |
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