曲安奈德胆管洗脱支架的制作及其在胆管良性狭窄中的应用
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摘要
目的
制作一种新型可以缓释曲安奈德的胆道支架，观察带药支架药物释放的规律,并评价其在兔胆管良性狭窄模型中应用的有效性及安全性。
方法
第一部分将曲安奈德和聚乳酸-羟基乙酸（PLGA）粉末以20%的带药浓度溶于两者的共同溶剂四氢呋喃（THF）中。并将6Fr胆道引流管浸剪成数个50mm段泡于上述溶剂中，10分钟后取出真空烘干，常温避光保存。通过测重计算支架所载曲安奈德的质量；采用磷酸盐缓冲液（PBS，PH7.4）将曲安奈德稀释为系列标准液，在240nm紫外分光峰面积进行相关分析。将曲安奈德支架放入8mlPBS中，置于37℃的恒温摇床中持续浸泡48h，然后换新鲜的PBS重复浸泡，直至第40天。以标准PBS液为对照组，对第1-40天留取的浸出液进行色谱分析，计算曲安奈德在浸出液中的浓度。
第二部分选用健康新西兰白兔36只，随机分3组，药物支架组（12只），普通支架组（12只）及对照组（12只）。电灼损伤胆总管随后经十二指肠乳头逆向插入支架至胆总管中,对照组仅电灼胆总管，无特殊处理，术后观察动物的一般状况、生存率，肝功能变化。30d后取狭窄处标本，HE染色光镜下观察胆管组织，免疫组织化学SP法测量TGF-β1及α-SMA的表达。
结果
第一部分称量测得平均每根支架上曲安奈德的负载量为510μg,单位面积的载药量为1.63μg/mm2。曲安奈德均能从支架表面持续释放，前5天浓度波动在6.32μg/ml到8.78μg/ml之间，前5天共释放曲安奈德180.61μg，占总释放量的47.23％，之后浓度平稳波动在1.85到3.51μg/ml之间，第23天后开始降低，第33天的洗脱浓度为0.53μg/ml，随后即不可测得。
第二部分植入药物及普通支架均可有效预防胆总管损伤后狭窄的形成，肝功能检测两支架组血清总胆红素无明显升高，药物支架组血清总胆红素波动在0.61μmol/l将至0.73μmol/l之间，普通支架组血清总胆红素波动在0.54μmol/l将至0.75μmol/l之间，对照组肝功能持续升高。病理学观察药物支架组胆管无纤维化增生，电灼处胆管直径较普通支架组略大。对照组电灼处管腔狭窄，炎性细胞浸润，黏膜下胶原纤维增生明显。免疫组化可见α-SMA、TGF-β1强表达于对照组狭窄胆管，在普通支架组电灼处胆管表达也较强，弱表达于药物支架组电灼处胆管，两支架组之间具有统计学意义（P＜0.05）。
结论
第一部分用PLGA作为药物载体能成功制备曲安奈德药物胆道洗脱支架；体外研究表明该药物洗脱支架可持续稳定释放曲安奈德超过30天。
第二部分曲安奈德药物支架在良性胆管狭窄中的应用是安全可行的，除了具有机械性扩张胆管的作用外，由于支架具有药物缓释作用，在局部可持续释放药物，对胆管瘢痕的形成起到一定的抑制作用。
Objective
Produce a new kind of biliary stent with Triamcinolone-eluting and observe the law ofdrug delivery from the stent evaluate the availability and safety in Biliary Benign Stricturemodel of rabbit
Methods
Part Ⅰ: The powder both the Triamcinolone and polylactic glycolic acid (PLGA)dissolved in the common solvent tetrahydrofuran (THF) with a drug concentration of20%.6F biliary soaked in the above solvents. After10minutes, remove the vacuum drying, andstored at room temperature. Calculated the quality of the stent contained in Triamcinoloneby measuring changes in the quality. PBS buffer (PH7.4) diluted Triamcinolone for thestandard solution and analyzed the ultraviolet peak area at240nm. Triamcinolone-elutingstents soaked in PBS, placed in shaker with a constant temperature of37°C continuingsoaked48h, and then soaked in fresh PBS solution until40days. Standard PBS solutionsas the control group, the chromatographic analysis of specimens from the leaching solutionfor1-40days, calculated the concentration of Triamcinolone in the leaching solution. PartⅡ:36New Zealand rabbits were randomly divided into Triamcinolone-Eluting Stentgroup1（n=12），polyurethane stent group2（n=12）and control group3（n=12）. Throughthe duodenal papilla reverse insert stent into the common bile duct after fulgurizecholedochus. Control group only fulgurize choledochus common bile duct withoutspecial treatment. one month later. General conditions, survival of the animals andChanges in liver function were observed after surgery.observe. the histological changes ofbile duct after30days; immunohistochemistry SP method was used to measuretransforming growth factor-β1(TGF-β1) and α-Smooth muscle actin(α-SMA) expression.
Results
Part Ⅰ: The quality of Triamcinolone contained in the stent was detected. Triamcinolone on the stent of capacity was up to510μg; per unit area contained in the drugamount was1.63μg/mm2. Triamcinolone could be sustained-release from the stent surface.The first five days the quality release from the stent was180.61μg，is47.23%of the totalamount release from the stent then it fluctuated within1.85-3.51μg/ml.The concentrationof Triamcinolone begane to decrease from the23st day，and it was0.53μg/ml on the33thday，it can’t be detected in the later days.
Part Ⅱ: Both of the two stent groups could effective prevent Biliary benige strictureformation after bile duct injuried.Stricture was improved in the two stent groups.Triamcinolone-Eluting Stent group total bilirubin fluctuate between0.61μmol/l and0.73μmol/l，polyurethane stent group total bilirubin fluctuate between0.54μmol/l and0.75μmol/l, there are no statistically significant between two stent groups(P＞0.05). totalbilirubin of the control group continued to rise. segment of the Stricture bile duct fromgroup1was expanded bigger than group2by histology observed. Inflammatory cellinfiltration, collagen fibers in the submucosal were observed from control group. Theimmunohistochemistry results showed that the stenosis bile duct of TGF-βl and α-SMAfrom group1expression were lower than group2,there have significant differencesbetween the two stent groups(P＜0.05).highest expression in the control group.
Conclusion
Part Ⅰ: Triamcinolone-eluting biliary stents can be successful prepared by polylacticglycolic acid as a drug carrier. In vitro study shows that the drug-eluting stents cansustainable and stable release of Triamcinolone over30days.
Part Ⅱ: the new Triamcinolone-Eluting Stent is safe and provides enhanced localdrug delivery.it can inhibition the form of Biliary scar to a certain degree.
制作一种新型可以缓释曲安奈德的胆道支架，观察带药支架药物释放的规律,并评价其在兔胆管良性狭窄模型中应用的有效性及安全性。
方法
第一部分将曲安奈德和聚乳酸-羟基乙酸（PLGA）粉末以20%的带药浓度溶于两者的共同溶剂四氢呋喃（THF）中。并将6Fr胆道引流管浸剪成数个50mm段泡于上述溶剂中，10分钟后取出真空烘干，常温避光保存。通过测重计算支架所载曲安奈德的质量；采用磷酸盐缓冲液（PBS，PH7.4）将曲安奈德稀释为系列标准液，在240nm紫外分光峰面积进行相关分析。将曲安奈德支架放入8mlPBS中，置于37℃的恒温摇床中持续浸泡48h，然后换新鲜的PBS重复浸泡，直至第40天。以标准PBS液为对照组，对第1-40天留取的浸出液进行色谱分析，计算曲安奈德在浸出液中的浓度。
第二部分选用健康新西兰白兔36只，随机分3组，药物支架组（12只），普通支架组（12只）及对照组（12只）。电灼损伤胆总管随后经十二指肠乳头逆向插入支架至胆总管中,对照组仅电灼胆总管，无特殊处理，术后观察动物的一般状况、生存率，肝功能变化。30d后取狭窄处标本，HE染色光镜下观察胆管组织，免疫组织化学SP法测量TGF-β1及α-SMA的表达。
结果
第一部分称量测得平均每根支架上曲安奈德的负载量为510μg,单位面积的载药量为1.63μg/mm2。曲安奈德均能从支架表面持续释放，前5天浓度波动在6.32μg/ml到8.78μg/ml之间，前5天共释放曲安奈德180.61μg，占总释放量的47.23％，之后浓度平稳波动在1.85到3.51μg/ml之间，第23天后开始降低，第33天的洗脱浓度为0.53μg/ml，随后即不可测得。
第二部分植入药物及普通支架均可有效预防胆总管损伤后狭窄的形成，肝功能检测两支架组血清总胆红素无明显升高，药物支架组血清总胆红素波动在0.61μmol/l将至0.73μmol/l之间，普通支架组血清总胆红素波动在0.54μmol/l将至0.75μmol/l之间，对照组肝功能持续升高。病理学观察药物支架组胆管无纤维化增生，电灼处胆管直径较普通支架组略大。对照组电灼处管腔狭窄，炎性细胞浸润，黏膜下胶原纤维增生明显。免疫组化可见α-SMA、TGF-β1强表达于对照组狭窄胆管，在普通支架组电灼处胆管表达也较强，弱表达于药物支架组电灼处胆管，两支架组之间具有统计学意义（P＜0.05）。
结论
第一部分用PLGA作为药物载体能成功制备曲安奈德药物胆道洗脱支架；体外研究表明该药物洗脱支架可持续稳定释放曲安奈德超过30天。
第二部分曲安奈德药物支架在良性胆管狭窄中的应用是安全可行的，除了具有机械性扩张胆管的作用外，由于支架具有药物缓释作用，在局部可持续释放药物，对胆管瘢痕的形成起到一定的抑制作用。
Objective
Produce a new kind of biliary stent with Triamcinolone-eluting and observe the law ofdrug delivery from the stent evaluate the availability and safety in Biliary Benign Stricturemodel of rabbit
Methods
Part Ⅰ: The powder both the Triamcinolone and polylactic glycolic acid (PLGA)dissolved in the common solvent tetrahydrofuran (THF) with a drug concentration of20%.6F biliary soaked in the above solvents. After10minutes, remove the vacuum drying, andstored at room temperature. Calculated the quality of the stent contained in Triamcinoloneby measuring changes in the quality. PBS buffer (PH7.4) diluted Triamcinolone for thestandard solution and analyzed the ultraviolet peak area at240nm. Triamcinolone-elutingstents soaked in PBS, placed in shaker with a constant temperature of37°C continuingsoaked48h, and then soaked in fresh PBS solution until40days. Standard PBS solutionsas the control group, the chromatographic analysis of specimens from the leaching solutionfor1-40days, calculated the concentration of Triamcinolone in the leaching solution. PartⅡ:36New Zealand rabbits were randomly divided into Triamcinolone-Eluting Stentgroup1（n=12），polyurethane stent group2（n=12）and control group3（n=12）. Throughthe duodenal papilla reverse insert stent into the common bile duct after fulgurizecholedochus. Control group only fulgurize choledochus common bile duct withoutspecial treatment. one month later. General conditions, survival of the animals andChanges in liver function were observed after surgery.observe. the histological changes ofbile duct after30days; immunohistochemistry SP method was used to measuretransforming growth factor-β1(TGF-β1) and α-Smooth muscle actin(α-SMA) expression.
Results
Part Ⅰ: The quality of Triamcinolone contained in the stent was detected. Triamcinolone on the stent of capacity was up to510μg; per unit area contained in the drugamount was1.63μg/mm2. Triamcinolone could be sustained-release from the stent surface.The first five days the quality release from the stent was180.61μg，is47.23%of the totalamount release from the stent then it fluctuated within1.85-3.51μg/ml.The concentrationof Triamcinolone begane to decrease from the23st day，and it was0.53μg/ml on the33thday，it can’t be detected in the later days.
Part Ⅱ: Both of the two stent groups could effective prevent Biliary benige strictureformation after bile duct injuried.Stricture was improved in the two stent groups.Triamcinolone-Eluting Stent group total bilirubin fluctuate between0.61μmol/l and0.73μmol/l，polyurethane stent group total bilirubin fluctuate between0.54μmol/l and0.75μmol/l, there are no statistically significant between two stent groups(P＞0.05). totalbilirubin of the control group continued to rise. segment of the Stricture bile duct fromgroup1was expanded bigger than group2by histology observed. Inflammatory cellinfiltration, collagen fibers in the submucosal were observed from control group. Theimmunohistochemistry results showed that the stenosis bile duct of TGF-βl and α-SMAfrom group1expression were lower than group2,there have significant differencesbetween the two stent groups(P＜0.05).highest expression in the control group.
Conclusion
Part Ⅰ: Triamcinolone-eluting biliary stents can be successful prepared by polylacticglycolic acid as a drug carrier. In vitro study shows that the drug-eluting stents cansustainable and stable release of Triamcinolone over30days.
Part Ⅱ: the new Triamcinolone-Eluting Stent is safe and provides enhanced localdrug delivery.it can inhibition the form of Biliary scar to a certain degree.
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