胆道内可降解支架的体内及体外的实验性研究
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摘要
目的：1、观察不同摩尔比的聚乳酸/羟基乙酸共聚物（PLGA）胆道支架在胆汁中降解速率和分子量的变化，从而筛选符合胆道塑形时间要求的聚乳酸/羟基乙酸共聚物（PLGA）来制造可生物降解的胆道支架。2、探讨胆总管探查术后内置可生物降解支架管在动物实验中的可行性，以增加胆管一期缝合的安全性，减少其并发症，扩展适应症的范围，在严格掌握适应症的前提下替代T管引流术。方法：1、选用不同摩尔比的聚乳酸/羟基乙酸共聚物（PLGA）50/50、60/40、70/30和80/20制作的可降解支架管，称取原始重量（w0），放入装有新鲜胆汁的安瓶中，置于37℃恒温培养箱，每日观测支架的形貌的变化，并更换胆汁。隔日取样本，清洗后，冻干并称量其干重（wt），观测30天，记录支架管的形貌变化，按公式(w0-wt)/w0×100%计算其失重率的变化，并测量降解后的分子量，分别绘制各自曲线。2、方法：用20只本地杂交犬（体重在20-25kg）模拟胆总管探查术，将其随机分为
两组：一期缝合组（对照组），胆总管内置可降解支架后行一期缝合组（实验组）。术前1天犬禁食，2.5%戊巴比妥钠（1ml/kg）静脉麻醉。游离胆总管，距十二指肠上缘2cm处纵行切开胆总管，切口长约5-8mm，置入已消毒的混有钡剂的可降解支架管，间断缝合，针距2mm、边距2mm。检查缝合口有无渗漏，于肝下放置胶管引流，术中输注0.9%生理盐水500ml加庆大霉素16万U，术后3日拔除引流管。观察指标：术中测量胆总管直径。术后有无胆漏。每周采血送检酶学检查；每周通过X线摄影观察引流管
的位置及形态，以及何时完全消失。当X线摄影下支架管显影完全消失时，将犬处死并取材，测量胆总管的直径，光镜下观察肝组织的变化。结果：1、（1）50/50 10天管体即出现裂纹，管腔完整无塌陷；15天后出现塌陷，裂解成碎片状。（2）60/40 12天管体出现纵纹，管壁变薄，18天出现管体碎裂。（3）70/30 14天管体出现细小纵纹，管壁变薄，20天出现片状碎裂。（4）80/20 20天管体出现细小纵纹，管壁变薄，管腔未见塌陷，到30天尚未发现管腔塌陷。失重曲线表明50/50和60/40配比的支架的降解高峰在6-10天，70/30和80/20的支架降解高峰在14-18天；2、对照组1只犬出现胆漏，实验组未出现胆漏；第5周时9只支架管体X线下显影完全消失，一只支架管仍然显影，第5周时9只支架管体X线下显影完全消失；仅1只还仍显影，解剖时发现支架管已碎裂并粘为一体，阻塞胆管，阻塞上方胆管明显扩张；对照组ALP、GPT、GOT明显升高，实验组无明显改变；对照组胆管及肝脏炎症轻微；胆总管直径，对照组明显狭窄；实验组轻微扩张。结论：1、70/30和80/20配比的PLGA支架符合胆道塑型时间的要求，降解时间符合T管外流的时间；2、胆总管探查术后内置可降解支架后再行胆管一期缝合安全可靠，较单纯一期缝合更加安全。在经过术中造影或探查确认无胆道残余结石、肝内胆管结石等的患者可以完全替代T形管，并能弥补胆道一期吻合术的局限性，此项技术具有广阔的发展前景。
Objective: 1、To study the degradation results of different molar ratio PLGA CBD stents in bile in vitro. To select one of them to replace the routine setting of T shape draining tube after CBDE. 2、To explore the possibility of inserting biodegradable instents after CBDE, in order to ensure the safety to the primary suture of CBD and allivate the complications, widen the scope of implications, and finally to replace the T tube drainage with strict implications.
Method: Degradable stents made of different molar ratio of LA/GA are stored in the vials containing fresh bile. Then incubate in 37 degree homeothermia incubator. To observe the change of shape, weight loss and molecular weight of stents. Sampling every two days, after cleaning it, when it was lyophilized, masure its weight. According to the formula (w0 wt)/w0×100% to observe the change of the rate of its weightlessness and measure molecular weight, then draw each curve. Use 20 local crossbreed dogs to mimic CBDE, randomized them into 2 groups: control group (not insert stents in bile duct, only primary suture), experimental group. Degradable stent was inserted in CBD with primary closure (experimental group). Evaluate parameters including: (1) CBD diameter. (2) bile leakage rate. (3) postoperative CBD diameter. (4) alkaline phosphatase activity (ALP), glutamic-pyruvic transaminase（GPT）, glutamic-oxalacetic transaminase (GOT). (5) When the developing
of the stent disappears under X-ray, sacrifice the dogs and compare the change of diameter of CBD with operation time. (6) Pathology of liver tissue.
Result:
1、The degradation peak times of 50/50 and 60/40 ration, 70/30 and 80/20 ration scaffold are after 6-10 days and 14-18 days, respectively.
2、There were no definitive statistical differences between two groups in bile leakage rate. One dog occurred bile leakage in comparative group, bile leakage was not found in experimental group.
3、There was only one stent remaining developing under X-ray, others were completely disappeared in the fifth week. We found the stent had already fragmentationed, and the fragments accumulated block the bile to go through fluently. The bile duct above the blocking area expanded accordingly.
4、In experimental group, dogs had no obvious changes in the level of ALP、GOT and GPT. However, in control group, dogs developed long-term high level of ALP, GOT and GPT.
5、The remarkable difference between two groups in postoperative CBD diameter and pathology of liver tissue showed: there was notable CBD obstruction in control group, but there was no temporary CBD obstruction in experimental group.
Conclusion: the degradation time of 70/30 and 80/20 ratio stents
is consistent with the termination time of T shape draining tube. We propose that the degradable stent placed in CBD with primary closure of it as a safe and effective technique adjunct to CBD exploration. Further assessment in a clinical trial is warranted. We firmly believe that if the new method is applied to patients, it will be associated with low morbidity and alleviate the complications of T-tubes.
两组：一期缝合组（对照组），胆总管内置可降解支架后行一期缝合组（实验组）。术前1天犬禁食，2.5%戊巴比妥钠（1ml/kg）静脉麻醉。游离胆总管，距十二指肠上缘2cm处纵行切开胆总管，切口长约5-8mm，置入已消毒的混有钡剂的可降解支架管，间断缝合，针距2mm、边距2mm。检查缝合口有无渗漏，于肝下放置胶管引流，术中输注0.9%生理盐水500ml加庆大霉素16万U，术后3日拔除引流管。观察指标：术中测量胆总管直径。术后有无胆漏。每周采血送检酶学检查；每周通过X线摄影观察引流管
的位置及形态，以及何时完全消失。当X线摄影下支架管显影完全消失时，将犬处死并取材，测量胆总管的直径，光镜下观察肝组织的变化。结果：1、（1）50/50 10天管体即出现裂纹，管腔完整无塌陷；15天后出现塌陷，裂解成碎片状。（2）60/40 12天管体出现纵纹，管壁变薄，18天出现管体碎裂。（3）70/30 14天管体出现细小纵纹，管壁变薄，20天出现片状碎裂。（4）80/20 20天管体出现细小纵纹，管壁变薄，管腔未见塌陷，到30天尚未发现管腔塌陷。失重曲线表明50/50和60/40配比的支架的降解高峰在6-10天，70/30和80/20的支架降解高峰在14-18天；2、对照组1只犬出现胆漏，实验组未出现胆漏；第5周时9只支架管体X线下显影完全消失，一只支架管仍然显影，第5周时9只支架管体X线下显影完全消失；仅1只还仍显影，解剖时发现支架管已碎裂并粘为一体，阻塞胆管，阻塞上方胆管明显扩张；对照组ALP、GPT、GOT明显升高，实验组无明显改变；对照组胆管及肝脏炎症轻微；胆总管直径，对照组明显狭窄；实验组轻微扩张。结论：1、70/30和80/20配比的PLGA支架符合胆道塑型时间的要求，降解时间符合T管外流的时间；2、胆总管探查术后内置可降解支架后再行胆管一期缝合安全可靠，较单纯一期缝合更加安全。在经过术中造影或探查确认无胆道残余结石、肝内胆管结石等的患者可以完全替代T形管，并能弥补胆道一期吻合术的局限性，此项技术具有广阔的发展前景。
Objective: 1、To study the degradation results of different molar ratio PLGA CBD stents in bile in vitro. To select one of them to replace the routine setting of T shape draining tube after CBDE. 2、To explore the possibility of inserting biodegradable instents after CBDE, in order to ensure the safety to the primary suture of CBD and allivate the complications, widen the scope of implications, and finally to replace the T tube drainage with strict implications.
Method: Degradable stents made of different molar ratio of LA/GA are stored in the vials containing fresh bile. Then incubate in 37 degree homeothermia incubator. To observe the change of shape, weight loss and molecular weight of stents. Sampling every two days, after cleaning it, when it was lyophilized, masure its weight. According to the formula (w0 wt)/w0×100% to observe the change of the rate of its weightlessness and measure molecular weight, then draw each curve. Use 20 local crossbreed dogs to mimic CBDE, randomized them into 2 groups: control group (not insert stents in bile duct, only primary suture), experimental group. Degradable stent was inserted in CBD with primary closure (experimental group). Evaluate parameters including: (1) CBD diameter. (2) bile leakage rate. (3) postoperative CBD diameter. (4) alkaline phosphatase activity (ALP), glutamic-pyruvic transaminase（GPT）, glutamic-oxalacetic transaminase (GOT). (5) When the developing
of the stent disappears under X-ray, sacrifice the dogs and compare the change of diameter of CBD with operation time. (6) Pathology of liver tissue.
Result:
1、The degradation peak times of 50/50 and 60/40 ration, 70/30 and 80/20 ration scaffold are after 6-10 days and 14-18 days, respectively.
2、There were no definitive statistical differences between two groups in bile leakage rate. One dog occurred bile leakage in comparative group, bile leakage was not found in experimental group.
3、There was only one stent remaining developing under X-ray, others were completely disappeared in the fifth week. We found the stent had already fragmentationed, and the fragments accumulated block the bile to go through fluently. The bile duct above the blocking area expanded accordingly.
4、In experimental group, dogs had no obvious changes in the level of ALP、GOT and GPT. However, in control group, dogs developed long-term high level of ALP, GOT and GPT.
5、The remarkable difference between two groups in postoperative CBD diameter and pathology of liver tissue showed: there was notable CBD obstruction in control group, but there was no temporary CBD obstruction in experimental group.
Conclusion: the degradation time of 70/30 and 80/20 ratio stents
is consistent with the termination time of T shape draining tube. We propose that the degradable stent placed in CBD with primary closure of it as a safe and effective technique adjunct to CBD exploration. Further assessment in a clinical trial is warranted. We firmly believe that if the new method is applied to patients, it will be associated with low morbidity and alleviate the complications of T-tubes.
引文
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Gharaibeh K, Heiss H. Biliary leakage following T-tube removal. Int. Surg, 2000;85:57-63.
Gillatt D, May R, Longstaff A. Complications of T-tube drainage of the common bile duct. Ann. R. Surg.Engl, 1985;67:307-1.
Horgan P, Campbell A, Gray G, Gillespie G. Biliary leakage and peritonitis following removal of T-tubes after bile duct exploration. Br. J. Surg, 1989;76:1297.
曹红. 腹腔镜经胆囊管胆道造影的临床价值. 第三军医大学学报, 2002;24(7):843-844.
李立波, 蔡秀军, 李君达, 等. 术中胆道造影在腹腔镜胆囊切除术中发现胆道损伤的作用.
Machi J, Sigel B. Operative ultrasound in general surgery. Am J Surg, 1996;172:15-20.
Krauss H, Kern E. Some current problems of biliary tract surgery: indications and technique of choledochotomy, intraoperative cholangiomanometry, primary closure of the common bile duct. Surgery, 1967 Dec;62(6):983-7.
Decker G, Borie F, Millat B, Berthou JC, Deleuze A, Drouard F, Guillon F, Rodier JG, Fingerhut A. One hundred laparoscopic choledochotomies with primary closure of the common bile duct. Surg Endosc, 2002 Oct 8.
Wu JS, Soper NJ. Comparison of laparoscopic choledochotomy closure techniques. Surg Endosc, 2002 Sep;16(9):1309-13.
Tu Z, Li J, Xin H, Zhu Q, Cai T. Primary choledochorrhaphy after common bile duct exploration. Dig Surg. 1999;16(2):137-9.
Seale AK, Ledet WP Jr. Primary common bile duct closure. Arch Surg. 1999 Jan.134(1):22-4.
Croce E, Golia M, Azzola M, Russo R, Crozzoli L, Olmi S, Pompa C, Borzio M. Laparoscopic choledochotomy with primary closure. Follow-up (5-44 months) of 31 patients. Surg Endosc 1996 Nov; 10(11):1064-8.
Sorensen VJ, Buck JR, Chung SK, Fath JJ, Horst HM, Obeid FN. Primary common bile duct closure following exploration: an effective alternative to routine biliatry drainage. Am Surg.1994 Jun; 60(6):451-4.
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