纳米银涂层抗菌塑料支架治疗胆漏实验研究
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摘要
背景与目的：胆漏(biliary leakage)常发生在胆道或胆道邻近脏器的损伤之后。跨过乳头括约肌将内支架置入胆管，可以降低胆管压力，减少胆汁外漏，从而达到治疗胆漏的目的。但支架置入胆管常见并发症是胆管炎、支架阻塞。早期的研究中发现，阻塞的支架中可以见到大量的细菌菌落。使用目前材料制作的支架及附件均无抗菌作用。研究证明低浓度的银离子对微生物具有杀灭作用。降低银颗粒的尺寸使其达到纳米级，可大大提高其抗菌活性。胆管纳米银涂层抗菌塑料支架的应用未见相关报道。本实验拟制备胆管纳米银涂层塑料支架，通过体外实验及动物实验探讨其抗菌性能及治疗胆漏效果，以期为胆漏治疗提供一种具有抗菌作用的新型支架，为其进一步临床应用提供实验依据。
材料和方法：1）通过化学氧化还原法，以胆管聚四氟乙烯支架为载体，硝酸银为原料，制备胆管纳米银涂层塑料支架，通过扫描电镜（SEM）、透射电镜（TEM）、X射线能谱分析(EDXA)等技术检测纳米银涂层微观形态和化学成分；通过扫描隧道显微镜（STM）检测纳米银涂层塑料胆管支架的弹性系数和摩擦系数；2）通过定性及定量实验探讨纳米银涂层塑料支架对肠道代表菌群大肠杆菌抗菌活性；采用SEM观察纳米银涂层塑料支架干预大肠杆菌粘附性能；3）8只健康巴马小型猪为研究对象，全麻下行腹腔镜手术，距胆囊管根部0.5-1.0cm处离断胆囊管，胆囊管残端开放，剥离切除胆囊；通过术前、术后24小时监测血常规、肝功；术后24小时行ERC；死亡动物及时解剖，存活动物术后48小时处死解剖观察胆囊管残端等方法综合判断腹腔镜下造模可行性；4）40只健康巴马小型猪用于研究。腹腔镜下建立胆漏动物模型，随机分为3组，内镜下猪胆管置入胆管纳米银涂层聚四氟乙烯支架（n=15)、普通胆管聚四氟乙烯支架（n=15)、对照组（造模后常规抗炎治疗，n=10）。及时解剖术后死亡动物，了解死亡原因。术前及术后随访抽血检测血常规、肝功；术后10天、20天随访处死各组1/2存活动物,通过透视了解支架位置，无菌操作取出支架，支架剖开进行细菌培养；通过SEM观察支架各段表面菌斑情况并比较；通过光学显微镜观察猪十二指肠乳头部、胆总管各段组织病理学变化并比较。
结果：1）成功制备胆管纳米银涂层聚四氟乙烯支架，纳米颗粒大小在5-80nm。纳米银涂层仅含有Ag；在一定范围内，纳米银涂层（0.5-5um）厚度增加，支架弹性系数（杨氏模量）增大，摩擦系数减小；2）定性抗菌实验：普通胆管支架周围没有形成抑菌圈；纳米银胆管支架周围可见有抑菌圈，直径为6.47±0.74mm。2组比较差异有统计学意义（p﹤0.05）；定量抗菌实验：长0.5cm，直径8.5Fr支架与106CFU/ml及104CFU/ml大肠杆菌菌悬液接触处后，随着培养时间的延长，实验组菌悬液（胆管纳米银涂层塑料支架）菌落数逐渐下降，接触6小时，106CFU/ml及104CFU/ml菌悬液中大肠杆菌被几乎全部被杀死，抗菌率达到100%；对照组菌悬液（普通胆管塑料支架）菌落数逐渐升高。SEM观察：胆管纳米银涂层塑料支架具有较强的干预大肠杆菌粘附作用；3）腹腔镜下能充分暴露胆囊、胆囊管、胆囊动脉，显示胆囊与肝脏及胆囊管与胆总管的解剖关系。与术前相比，术后24小时白细胞、中性粒细胞、直接胆红素明显升高（p﹤0.05）。8只实验猪7只距胆囊管根部0.5-1.0cm处离断胆囊管，1只距胆囊管根部约2cm处离断胆囊管；8只实验猪行ERC,7只造影剂明显外漏,1只造影剂外漏不明显；4）40头实验猪成功造模、30只内镜下置入支架，10只对照观察。支架组3只实验猪术后20-38小时内死亡，解剖死亡原因为胆总管穿孔。对照组10只术后5天内全部死亡，死亡原因胆汁性腹膜炎伴感染。对照组死亡率与2支架组比较差异均有统计学意义（p﹤0.05）；术后10天，2支架组白细胞水平均较术前明显增高（p﹤0.05），术后20天，普通支架组白细胞水平较术前明显增高（p﹤0.05），纳米银支架组较术前无明显变化（p＞0.05）；术后10天、20天，所有实验猪支架内容物细菌培养菌均阳性，比较无差异（p＞0.05）；2组均出现胆总管穿孔，支架异位，比较差异无统计学意义（p＞0.05）；扫描电镜观察，纳米银支架及普通支架表面均有数量不等的炎性附着物或结晶体黏附在支架表面，细菌分布其中。纳米银支架表面粘附细菌明显少于普通支架。组内比较，下段胆管支架表面粘附细菌较中段、上段胆管支架多；光学显微镜观察猪十二指肠乳头部、胆总管各段组织病理学变化，发现纳米银支架组、普通支架组胆管粘膜上皮不同程度缺失，壁内可见血管扩张充血，炎细胞浸润。但普通支架组胆管全层炎细胞浸润更为明显，部分切片可见脓肿形成。
结论：1）通过化学氧化还原法，以聚四氟乙烯塑料胆管支架为载体，硝酸银为原料，制备胆管纳米银涂层塑料支架各项理化性能良好；2）纳米银涂层胆管支架对大肠杆菌具有较强抗菌作用和干预粘附作用；3）腹腔镜下制备猪胆漏动物模型安全、可行，成功率高；4）进一步证实内镜下胆管内置入塑料支架可以治疗胆漏；体内实验，胆管纳米银涂层塑料支架具有干扰表面细菌粘附，抑制细菌生物被膜形成作用；对局部胆管炎症发生具有明显抑制作用，促进胆漏愈合。
Background and objective：Biliary leakage often happened after the injury of thebiliary tract or the organs nearby. Insertion of endoprosthesis into the bilary tract throughthe sphincter of the duodenal papilla can lower the pressure of the biliary tract, and thenreduce leakage of the bile,which aims to treat biliary leakage. But the commoncomplications of the endoprosthesis are occlusion of the stents, and cholangitis. Some paststudies have shown that there are many bacterial colonies in the blocked stents and thestents made of materials usually used now and the attachment have no antibacterial effects.However, some studies have proved that the silver in a low concentration has theantimicrobial efficacy and the decrease of the the silver’s size to nano particles can greatlyincrease its antibacterial effectiveness. By now, there are no reports about the applicationof the endoprosthesis coated with nano-silver. Therefore, plastic stents coated withnano-silver are prepared and then investigated their antimicrobial properties andeffectiveness of treating biliary leakage through the animal experiment and in vitro test forthe purpose of providing a new endoprosthesis for the biliary leakage and a base for thefurther clinical experiments.
Methods:1) The plastic biliary stents coated with nano-silver were prepared by thechemical redox process with teflon plastic stents as carriers and silver nitrate as material.Then the micromorphology and chemical composition of the coat were dectected by use ofscanning electron microscope（SEM）,transmission electron microscopy （TEM）andEnergy Dispersive X-ray analysis（EDXA）, and the elastic modulus and the frictionalcoefficient of the stents were measured by scanning tunneling microscope（STM）.2) Theantibacterial properties of the plastic biliary stents coated with nano-silver to colibacillus,the dominant flora in the intestines were studied qualitatively and quantitatively.Meantime,their effects of antiadhesion to the colibacillus were observed by SEM.3)8healthy Bama Minipigs were used as the objects and underwent laparoscopic surgery under general anesthesia.The cystic duct was broken at0.5-1.0cm away from its root and thestump was left open. Finally the gallbladder was separated and removed. Then thefeasibility of these models was judged according to the following aspects: blood routineand hepatic function were monitored in24h pre-and post surgery; endoscopic retrogradecholangiography (ERC) was performed at24h after the surgery; the dead pigs in themidway were butchered immediately and the survival pigs at48hours after the surgerywere butchered for observation the stump of the cystic duct.4)40standard Bama minipigswere included in this study. They were established as animal models of biliary leakagethrough laparoscopic surgeries and then divided into3groups randomly. The biliary stentscoated with nano-silver were used in one group(n=15), the common stents in anothergroup(n=15)，and the routine anti-inflammatory treatment was given to the control group(n=10). The accidental dead pigs were butchered immediately for the cause of death. Bloodrountine,hepatic function and blood culture were detected regularly preoperation andpostoperation. After the following-up of10days and20days, the survival animals werebutchered half in every group separately. The position of the stents was observed byfluoroscopy. Then the stents were removed out under the sterile condition and the contentswere used to bacterial culture. The bacteria plaques on the suface of the3sections werecompared under SEM. The pathological changes in duodenal papilla,the3segment of thecommon bile ducts were observed and compared by light microscope.
Results:1)The teflon stents coated with nano-silver were prepared successfully andthe size of silver particles was from5to80nm. The coat only contained argentum withoutother chemical constituents.With the increasing of thickness of the coat (0.5-5um),theelastic modulus (Young 's modulus) increased and the frictional coefficient decreased in acertain range.2）Qualitative antibacterial tests showed that there were no inhibition zonearound the common biliary stents, while around the stents coated with nano-ailver,therewere inhibition zones with dimatere6.47±0.74mm, which made a significant differencebetween the two group（sp﹤0.05）.Quantitive antibacterial tests showed that after the stentswith length0.5cm and diameter8.5Fr were put into two different concentrations ofcolibacillary suspension(106CFU/ml and104CFU/ml), with the increasing of the culture time, the number of bacterial colonies decreased in the experimental group (with stentscoated with nano-silver) and increased in the control group (with common stents); thecolibacillus was killed totally after6hours of culture in the later concentration and theantibacterial rate was100%; the stents coated with nano-silver performed a strongerantiadhesive activity on the colibacillus by SEM.3)The gallbladder, cystic duct,cysticartery and the anatomical relation between the gallbladder and liver together with thecystic duct and commom bile duct could be fully exposed under laparoscopy. Compared topreoperation, the white blood cells,neutrophil and direct bilirubin increased higher afterthe operation（p﹤0.05）. Among the8experimental pigs, the cystic ducts of7ones werebroken at0.5-1cm away from the roots of the cystic ducts and one at2cm. ERC wasperformed in8pigs, among which7ones were found obvious leakage of the contrastmedia and one was not.4)Among40minipigs successfully established as the models,30ones underwent the endoprosthesis and the other10ones did not as the control.3pigs inthe stents’group died within20-38hours after the surgery because of the perforation of thecommon bile ducts,which was proved after butchered. All10ones in the control groupdied with5days after surgery because of biliary peritonitis together with infection. Themortality between the stent group and the control group was significantly different（p﹤0.05）. Compared to preoperation, the white blood cells in the stent group increasedhigher after the following-up10days（p﹤0.05）and that in the common stent groupincreased higher after the follow-up20day（p﹤0.05）,while that in the group with stentscoated with nano-silver were not found obvisous increase. The contents of the stents werecultured and the bacteria were positive in all pigs after the following-up10days and20days without significant difference in two different stent groups（p＞0.05）. The two kindsof stents both caused perforation, migrated and slided out，which showed no significantdifferences between them（p＞0.05）. On the suface of the stents in the two groups, therewere varying amounts of inflammatory attachments or cystals adhered and bacteria alsodistributed among them. The number of the bacteria in the stents coated with nano-silvewas significantly lower than that in the common stents. The inferior segment of the stentshad more bacteria than the superior and middle part in both two kinds of stents. By light microscope, the pathological changes in duodenal papilla, the equal3segments of thecommon bile ducts were described as follows: mucosal epithelium missed in differentdegrees, the blood vessels in the mucosal layer were dilated and hyperemia, and theinflammatory cells infiltrated in both two stent groups; what’ more, in the common stentgroup, the infiltrating of the inflammatory cells within the whole layer was more apparentand abscess could be seen in some pathological sections.
Conclusions:1)The plastic biliary stents coated with nano-sliver are prepared by thechemical redox process with teflon plastic stents as carriers and silver nitrate as material.and perform good physical and chemical properties.2）The biliary stents coated withnano-sliver have strong antibacterial and antiadhesive effects on colibacillus.3)Themodels of biliary leakage established under laparoscopy are safe and feasible with a highsuccessful rate;4)Biliary leakage can be treated by inserting plastic stents into biliary tractsof the minipigs. The following up after the endoprosthesis shows that the stents coatedwith nano-silver can interfere with bacterial adhesion and the formation of bacterialbiomembranes and have obvisous inhibitory actions on local cholangitis to promote thehealing of biliary leakage.
材料和方法：1）通过化学氧化还原法，以胆管聚四氟乙烯支架为载体，硝酸银为原料，制备胆管纳米银涂层塑料支架，通过扫描电镜（SEM）、透射电镜（TEM）、X射线能谱分析(EDXA)等技术检测纳米银涂层微观形态和化学成分；通过扫描隧道显微镜（STM）检测纳米银涂层塑料胆管支架的弹性系数和摩擦系数；2）通过定性及定量实验探讨纳米银涂层塑料支架对肠道代表菌群大肠杆菌抗菌活性；采用SEM观察纳米银涂层塑料支架干预大肠杆菌粘附性能；3）8只健康巴马小型猪为研究对象，全麻下行腹腔镜手术，距胆囊管根部0.5-1.0cm处离断胆囊管，胆囊管残端开放，剥离切除胆囊；通过术前、术后24小时监测血常规、肝功；术后24小时行ERC；死亡动物及时解剖，存活动物术后48小时处死解剖观察胆囊管残端等方法综合判断腹腔镜下造模可行性；4）40只健康巴马小型猪用于研究。腹腔镜下建立胆漏动物模型，随机分为3组，内镜下猪胆管置入胆管纳米银涂层聚四氟乙烯支架（n=15)、普通胆管聚四氟乙烯支架（n=15)、对照组（造模后常规抗炎治疗，n=10）。及时解剖术后死亡动物，了解死亡原因。术前及术后随访抽血检测血常规、肝功；术后10天、20天随访处死各组1/2存活动物,通过透视了解支架位置，无菌操作取出支架，支架剖开进行细菌培养；通过SEM观察支架各段表面菌斑情况并比较；通过光学显微镜观察猪十二指肠乳头部、胆总管各段组织病理学变化并比较。
结果：1）成功制备胆管纳米银涂层聚四氟乙烯支架，纳米颗粒大小在5-80nm。纳米银涂层仅含有Ag；在一定范围内，纳米银涂层（0.5-5um）厚度增加，支架弹性系数（杨氏模量）增大，摩擦系数减小；2）定性抗菌实验：普通胆管支架周围没有形成抑菌圈；纳米银胆管支架周围可见有抑菌圈，直径为6.47±0.74mm。2组比较差异有统计学意义（p﹤0.05）；定量抗菌实验：长0.5cm，直径8.5Fr支架与106CFU/ml及104CFU/ml大肠杆菌菌悬液接触处后，随着培养时间的延长，实验组菌悬液（胆管纳米银涂层塑料支架）菌落数逐渐下降，接触6小时，106CFU/ml及104CFU/ml菌悬液中大肠杆菌被几乎全部被杀死，抗菌率达到100%；对照组菌悬液（普通胆管塑料支架）菌落数逐渐升高。SEM观察：胆管纳米银涂层塑料支架具有较强的干预大肠杆菌粘附作用；3）腹腔镜下能充分暴露胆囊、胆囊管、胆囊动脉，显示胆囊与肝脏及胆囊管与胆总管的解剖关系。与术前相比，术后24小时白细胞、中性粒细胞、直接胆红素明显升高（p﹤0.05）。8只实验猪7只距胆囊管根部0.5-1.0cm处离断胆囊管，1只距胆囊管根部约2cm处离断胆囊管；8只实验猪行ERC,7只造影剂明显外漏,1只造影剂外漏不明显；4）40头实验猪成功造模、30只内镜下置入支架，10只对照观察。支架组3只实验猪术后20-38小时内死亡，解剖死亡原因为胆总管穿孔。对照组10只术后5天内全部死亡，死亡原因胆汁性腹膜炎伴感染。对照组死亡率与2支架组比较差异均有统计学意义（p﹤0.05）；术后10天，2支架组白细胞水平均较术前明显增高（p﹤0.05），术后20天，普通支架组白细胞水平较术前明显增高（p﹤0.05），纳米银支架组较术前无明显变化（p＞0.05）；术后10天、20天，所有实验猪支架内容物细菌培养菌均阳性，比较无差异（p＞0.05）；2组均出现胆总管穿孔，支架异位，比较差异无统计学意义（p＞0.05）；扫描电镜观察，纳米银支架及普通支架表面均有数量不等的炎性附着物或结晶体黏附在支架表面，细菌分布其中。纳米银支架表面粘附细菌明显少于普通支架。组内比较，下段胆管支架表面粘附细菌较中段、上段胆管支架多；光学显微镜观察猪十二指肠乳头部、胆总管各段组织病理学变化，发现纳米银支架组、普通支架组胆管粘膜上皮不同程度缺失，壁内可见血管扩张充血，炎细胞浸润。但普通支架组胆管全层炎细胞浸润更为明显，部分切片可见脓肿形成。
结论：1）通过化学氧化还原法，以聚四氟乙烯塑料胆管支架为载体，硝酸银为原料，制备胆管纳米银涂层塑料支架各项理化性能良好；2）纳米银涂层胆管支架对大肠杆菌具有较强抗菌作用和干预粘附作用；3）腹腔镜下制备猪胆漏动物模型安全、可行，成功率高；4）进一步证实内镜下胆管内置入塑料支架可以治疗胆漏；体内实验，胆管纳米银涂层塑料支架具有干扰表面细菌粘附，抑制细菌生物被膜形成作用；对局部胆管炎症发生具有明显抑制作用，促进胆漏愈合。
Background and objective：Biliary leakage often happened after the injury of thebiliary tract or the organs nearby. Insertion of endoprosthesis into the bilary tract throughthe sphincter of the duodenal papilla can lower the pressure of the biliary tract, and thenreduce leakage of the bile,which aims to treat biliary leakage. But the commoncomplications of the endoprosthesis are occlusion of the stents, and cholangitis. Some paststudies have shown that there are many bacterial colonies in the blocked stents and thestents made of materials usually used now and the attachment have no antibacterial effects.However, some studies have proved that the silver in a low concentration has theantimicrobial efficacy and the decrease of the the silver’s size to nano particles can greatlyincrease its antibacterial effectiveness. By now, there are no reports about the applicationof the endoprosthesis coated with nano-silver. Therefore, plastic stents coated withnano-silver are prepared and then investigated their antimicrobial properties andeffectiveness of treating biliary leakage through the animal experiment and in vitro test forthe purpose of providing a new endoprosthesis for the biliary leakage and a base for thefurther clinical experiments.
Methods:1) The plastic biliary stents coated with nano-silver were prepared by thechemical redox process with teflon plastic stents as carriers and silver nitrate as material.Then the micromorphology and chemical composition of the coat were dectected by use ofscanning electron microscope（SEM）,transmission electron microscopy （TEM）andEnergy Dispersive X-ray analysis（EDXA）, and the elastic modulus and the frictionalcoefficient of the stents were measured by scanning tunneling microscope（STM）.2) Theantibacterial properties of the plastic biliary stents coated with nano-silver to colibacillus,the dominant flora in the intestines were studied qualitatively and quantitatively.Meantime,their effects of antiadhesion to the colibacillus were observed by SEM.3)8healthy Bama Minipigs were used as the objects and underwent laparoscopic surgery under general anesthesia.The cystic duct was broken at0.5-1.0cm away from its root and thestump was left open. Finally the gallbladder was separated and removed. Then thefeasibility of these models was judged according to the following aspects: blood routineand hepatic function were monitored in24h pre-and post surgery; endoscopic retrogradecholangiography (ERC) was performed at24h after the surgery; the dead pigs in themidway were butchered immediately and the survival pigs at48hours after the surgerywere butchered for observation the stump of the cystic duct.4)40standard Bama minipigswere included in this study. They were established as animal models of biliary leakagethrough laparoscopic surgeries and then divided into3groups randomly. The biliary stentscoated with nano-silver were used in one group(n=15), the common stents in anothergroup(n=15)，and the routine anti-inflammatory treatment was given to the control group(n=10). The accidental dead pigs were butchered immediately for the cause of death. Bloodrountine,hepatic function and blood culture were detected regularly preoperation andpostoperation. After the following-up of10days and20days, the survival animals werebutchered half in every group separately. The position of the stents was observed byfluoroscopy. Then the stents were removed out under the sterile condition and the contentswere used to bacterial culture. The bacteria plaques on the suface of the3sections werecompared under SEM. The pathological changes in duodenal papilla,the3segment of thecommon bile ducts were observed and compared by light microscope.
Results:1)The teflon stents coated with nano-silver were prepared successfully andthe size of silver particles was from5to80nm. The coat only contained argentum withoutother chemical constituents.With the increasing of thickness of the coat (0.5-5um),theelastic modulus (Young 's modulus) increased and the frictional coefficient decreased in acertain range.2）Qualitative antibacterial tests showed that there were no inhibition zonearound the common biliary stents, while around the stents coated with nano-ailver,therewere inhibition zones with dimatere6.47±0.74mm, which made a significant differencebetween the two group（sp﹤0.05）.Quantitive antibacterial tests showed that after the stentswith length0.5cm and diameter8.5Fr were put into two different concentrations ofcolibacillary suspension(106CFU/ml and104CFU/ml), with the increasing of the culture time, the number of bacterial colonies decreased in the experimental group (with stentscoated with nano-silver) and increased in the control group (with common stents); thecolibacillus was killed totally after6hours of culture in the later concentration and theantibacterial rate was100%; the stents coated with nano-silver performed a strongerantiadhesive activity on the colibacillus by SEM.3)The gallbladder, cystic duct,cysticartery and the anatomical relation between the gallbladder and liver together with thecystic duct and commom bile duct could be fully exposed under laparoscopy. Compared topreoperation, the white blood cells,neutrophil and direct bilirubin increased higher afterthe operation（p﹤0.05）. Among the8experimental pigs, the cystic ducts of7ones werebroken at0.5-1cm away from the roots of the cystic ducts and one at2cm. ERC wasperformed in8pigs, among which7ones were found obvious leakage of the contrastmedia and one was not.4)Among40minipigs successfully established as the models,30ones underwent the endoprosthesis and the other10ones did not as the control.3pigs inthe stents’group died within20-38hours after the surgery because of the perforation of thecommon bile ducts,which was proved after butchered. All10ones in the control groupdied with5days after surgery because of biliary peritonitis together with infection. Themortality between the stent group and the control group was significantly different（p﹤0.05）. Compared to preoperation, the white blood cells in the stent group increasedhigher after the following-up10days（p﹤0.05）and that in the common stent groupincreased higher after the follow-up20day（p﹤0.05）,while that in the group with stentscoated with nano-silver were not found obvisous increase. The contents of the stents werecultured and the bacteria were positive in all pigs after the following-up10days and20days without significant difference in two different stent groups（p＞0.05）. The two kindsof stents both caused perforation, migrated and slided out，which showed no significantdifferences between them（p＞0.05）. On the suface of the stents in the two groups, therewere varying amounts of inflammatory attachments or cystals adhered and bacteria alsodistributed among them. The number of the bacteria in the stents coated with nano-silvewas significantly lower than that in the common stents. The inferior segment of the stentshad more bacteria than the superior and middle part in both two kinds of stents. By light microscope, the pathological changes in duodenal papilla, the equal3segments of thecommon bile ducts were described as follows: mucosal epithelium missed in differentdegrees, the blood vessels in the mucosal layer were dilated and hyperemia, and theinflammatory cells infiltrated in both two stent groups; what’ more, in the common stentgroup, the infiltrating of the inflammatory cells within the whole layer was more apparentand abscess could be seen in some pathological sections.
Conclusions:1)The plastic biliary stents coated with nano-sliver are prepared by thechemical redox process with teflon plastic stents as carriers and silver nitrate as material.and perform good physical and chemical properties.2）The biliary stents coated withnano-sliver have strong antibacterial and antiadhesive effects on colibacillus.3)Themodels of biliary leakage established under laparoscopy are safe and feasible with a highsuccessful rate;4)Biliary leakage can be treated by inserting plastic stents into biliary tractsof the minipigs. The following up after the endoprosthesis shows that the stents coatedwith nano-silver can interfere with bacterial adhesion and the formation of bacterialbiomembranes and have obvisous inhibitory actions on local cholangitis to promote thehealing of biliary leakage.
引文
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