CA/PEI亲和膜去除肝硬化腹水中内毒素的研究
摘要
背景:目前腹水浓缩(或超滤)回输被认为是肝硬化失代偿期顽固性腹水的有效治疗方法之一。腹水的直接回输、浓缩回输或采用特定孔径的半透膜所进行的超滤回输,迅速消除了腹水的同时又回输了腹水中的蛋白质,但是腹水中的内毒素、胆红素等有毒物质也被回输到患者体内。二醋酸纤维素(CA)/聚乙烯基亚胺(PEI)共混制备微孔滤膜(以下简称CA/PEI亲和膜)能有效去除多种溶液中的内毒素,对胆红素也有吸附作用。CA/PEI亲和膜能否有效去除腹水中的内毒素,满足腹水回输治疗的需要,是本课题研究的出发点。
目的:本实验对CA/PEI亲和膜去除肝硬化腹水中的内毒素进行了研究。考察了流速、溶液中蛋白质浓度、温度等因素对去除腹水中内毒素效果的影响,并对其结果作了分析,期望为肝硬化顽固性腹水的回输治疗提供一个安全有效的措施。
方法:采用静态吸附和动态吸附两种方法,在不同条件(流速、溶液中蛋白质浓度、温度等)下对腹水中内毒素去除效果进行了研究,同时也考察了CA/PEI亲和膜对腹水中胆红素的吸附情况。应用BET-16细菌内毒素测定仪,采用动态浊度法,选择灵敏度为0.06EU/ml的鲎试剂,测定内毒素浓度,同时在我院化验室应用双缩脲法测定腹水中蛋白浓度及钒酸盐法测定腹水中胆红素浓度,根据吸附前后的浓度差,计算内毒素及胆红素去除率,蛋白回收率。
结果:1.亲和膜对无人血白蛋白(HSA)水溶液中内毒素的去除率,静态法89.1%,动态法98.5%;加入HSA水溶液中内毒素的去除率,静态法82.0%,动态法87.8%。可见蛋白质影响了去离子水溶液中内毒素的去除。蛋白回收率,静态法98.0%,动态法96.1%,说明亲和膜对HSA吸附性很小。温度在4℃、25℃、37℃条件下去离子水溶液中内毒素
Background:At present, there is still no operative method for the treatment of refractory ascites of liver cirrhosis. Direct or concentrated reinfusion of self-ascites as well as ultrafiltration ascites reinfusion by definite objective bapertur dialyzer can not only preclud the ascites quickly but also get the albumin in the ascites back into the body. However the noxious substance such as endotoxin and bilirubin will convey back the circulation at the same time. The microfiltration membrane adsorber which can selectively remove endotoxin by employing polyethyleneimine (PEI)as affinity ligand, was prepared by blending cellulose diacetate(CA) and PEI. CA/PEI affinity memberane has achieved efficient adsorptioin of endotoxin and bilirubin from some goal solutes. We hope this membrane can meet the demands in the application of ascites reinfusion treatment .
Objective:This study mainly deal with the removal of endotoxin in the ascites of liver cirrhosis by CA/PEI memberane. In order to get the appropriate condition for the adsorption of endotoxin, the ascites flow rate, the temperature and the concentration of protein were inspected and analyzed. We expected to provide a new material in the ascites reinfusion treatment which can effectively get rid of endotoxin before the ascites being concentrated or ultrafiltrated and then going back into the patients.
Methods: Through static and dynamic adsorption using CA/PEI memberane, we investigated and compared the effects on deionized water and the ascites endotoxin removal in different conditions(flow rate, protein concentration and temperature). we also simultaneously paid attention to the removal of bilirubin in the ascites. Kinetic-Turbidimetric methods was
目的:本实验对CA/PEI亲和膜去除肝硬化腹水中的内毒素进行了研究。考察了流速、溶液中蛋白质浓度、温度等因素对去除腹水中内毒素效果的影响,并对其结果作了分析,期望为肝硬化顽固性腹水的回输治疗提供一个安全有效的措施。
方法:采用静态吸附和动态吸附两种方法,在不同条件(流速、溶液中蛋白质浓度、温度等)下对腹水中内毒素去除效果进行了研究,同时也考察了CA/PEI亲和膜对腹水中胆红素的吸附情况。应用BET-16细菌内毒素测定仪,采用动态浊度法,选择灵敏度为0.06EU/ml的鲎试剂,测定内毒素浓度,同时在我院化验室应用双缩脲法测定腹水中蛋白浓度及钒酸盐法测定腹水中胆红素浓度,根据吸附前后的浓度差,计算内毒素及胆红素去除率,蛋白回收率。
结果:1.亲和膜对无人血白蛋白(HSA)水溶液中内毒素的去除率,静态法89.1%,动态法98.5%;加入HSA水溶液中内毒素的去除率,静态法82.0%,动态法87.8%。可见蛋白质影响了去离子水溶液中内毒素的去除。蛋白回收率,静态法98.0%,动态法96.1%,说明亲和膜对HSA吸附性很小。温度在4℃、25℃、37℃条件下去离子水溶液中内毒素
Background:At present, there is still no operative method for the treatment of refractory ascites of liver cirrhosis. Direct or concentrated reinfusion of self-ascites as well as ultrafiltration ascites reinfusion by definite objective bapertur dialyzer can not only preclud the ascites quickly but also get the albumin in the ascites back into the body. However the noxious substance such as endotoxin and bilirubin will convey back the circulation at the same time. The microfiltration membrane adsorber which can selectively remove endotoxin by employing polyethyleneimine (PEI)as affinity ligand, was prepared by blending cellulose diacetate(CA) and PEI. CA/PEI affinity memberane has achieved efficient adsorptioin of endotoxin and bilirubin from some goal solutes. We hope this membrane can meet the demands in the application of ascites reinfusion treatment .
Objective:This study mainly deal with the removal of endotoxin in the ascites of liver cirrhosis by CA/PEI memberane. In order to get the appropriate condition for the adsorption of endotoxin, the ascites flow rate, the temperature and the concentration of protein were inspected and analyzed. We expected to provide a new material in the ascites reinfusion treatment which can effectively get rid of endotoxin before the ascites being concentrated or ultrafiltrated and then going back into the patients.
Methods: Through static and dynamic adsorption using CA/PEI memberane, we investigated and compared the effects on deionized water and the ascites endotoxin removal in different conditions(flow rate, protein concentration and temperature). we also simultaneously paid attention to the removal of bilirubin in the ascites. Kinetic-Turbidimetric methods was
引文
1. 张顺财主编.内毒素基础与临床.北京,科学出版社,2003,1-21.
2. 李春梅,黄天卫,傅永潜.肝硬化患者内毒素测定的临床意义.实用内科学杂志,1988,8(1): 294-295.
3. 刘天舒,陈世耀,蔡鸿菊,等.自身腹水浓缩回输治疗难治性腹水的回顾性分析.中国临床医学,2003,10(2):164-166.
4. 朱宁川,陈岩.肝病肠道菌群失调与肠源性内毒素血症.中国微生态学杂志,2004,16(1): 61-62.
5. 周 喜 汉 . 肝 硬 化 顽 固 性 腹 水 治 疗 进 展 . 右 江 民 族 医 学 院 学报,2003,25(4):564-565.
6. 陈兆安.CA/PE 微孔亲和膜的制备及其性能与应用的研究.中国科学院大连化学物理研究所博士论文,2004.
7. A. Bruil, Leukocyte filters: Filtration mechanisms and material design. 1993,15-08.
8. D.Petsch, W.-D.Dechwer, F.B.Anspach, et al. Endotoxin removal with poly(ethyleneimine)-immobilized adsorbers: Sepharose 4B versus flat sheet and hollow fibre membranes. J.Chtomatogr.B,1998,707:121-130.
9. D.Petsch,T.C. Beeskow, F.B.Anspach, et al .Membrane adsorbers for selective removal of bacterisl endotoxin.J.Chtogr.B, 1997,693:79-91.
10. 中华人民共和国药典.2005 年版,二部,化学工业出版社.
11. 王思理主编.细菌内毒素检查法-定量法.
12. 魏桂林.亲和膜用于医药及体液中内毒素等的去除研究.中国科学院大连化学物理研究所硕士论文,2000.
13 D.Petsch,F.B.Anspach, Endotoxin removal from protein solutions.J. Biotech,2000,76:97-119.
14. Y.Kang,RG.Luo.Effects of ionic strength and PH on endotoxin removal Efficxiency and protein recovery in an affinity chromatography. Proc.Biochem,2000,36:85-92.
15. 商振华,周冬梅,郭为,等.聚酰胺亲和膜用于去除内毒素的研究.分析测试学报,1998,17(2):8-10.
16. 魏桂林,商振华,于亿年,等.新型亲和膜色谱用于去除胆红素的研究.色谱,2001,19(1):74-77.
1. 罗海波,朱平,李兰娟,等编.细菌毒素与临床[M].北京,人民卫生出版社,1999,9-10.
2. 赵龙凤,韩德武.肝病患者内毒素血症的临床意义.世界华人消化杂志,1999.7(5):391.
3. 姚咏明,张立天,陆家齐,等.脓毒症大鼠内毒素增敏系统改变与高迁移率族蛋白-1 表达的关系.中华创伤杂志,2002,18(9):540-543.
4. 唐虹.内毒素及内毒素血症治疗研究进展.医学综述,2005,11(2):109-110.
5. 中村东树他.肝病洞壁细胞研究の进展.东京,国际医书出版,1990,248.
6. Fox ES, Thomas,Broitman SA. The macrophage 80 kda endotoxin binding protein is not present on rat kupffer cells . Gastroenterology, 1990,98:586.
7. Shakamoto W,Nishihiral, Fujie K, et al.[J].Nutr Sci Vitaminol(Tokyo),2000Dec, 46(6):316.
8. 王顺宾.抗生素诱导革兰阴性杆菌释放内毒素的研究进展.国外医药抗生素分册,1998, 19(5):342-343.
9. Bucklin SE,Lake P, Logdberg L, et al. Therapeutic efficacy of a polymyxin B-dextran 70 conjugate in experimental model of endotoxemia. Antimicro Agens chemother.1995, 39(7):1462-1466.
10. 陆付耳,李鸣真,叶望云,等.话内毒素血症的治疗对策.中国危重病危急救医学,2000, 12(10):579.
11. 梅 受 敏 , 张 淑 慧 . 治 疗 肝 病 内 毒 素 血 症 进 展 . 中 国 医 院 药 学 杂志,2003,23(2):111-112.
12. 冯俊明,史景泉,刘友生.脂多糖对枯否细胞中 CD14, Toll 样受体 4 表达的影响及其意义.中华实验外科杂志,2002,19(4):350-352.
13. 楼福乐,毛伟钢,陆晓峰,等.超滤技术在制药工业中除热原的应用.膜科学与技术,1999, 19(3):8-12.
14. 龚承元,苏建勇,朱孟府.高电微孔滤膜的制备及其性能的初步研究.中国药学杂志,1994, 29(4):214-217.
15. 苏建勇,龚承元,朱孟府.高电微孔滤膜去除细菌内毒素的试验研究.军事医学科学院院刊,1995,19(4):289-292.
16. Howard F,Spooner ECR.Removal of taints from water . chem Ind,1946,186-189.
17. Statoshi Minobe,Taizo Watanabe,Tadashi Sato,et al.Characteristics and applications of adsorbents for pyrogen remova.Biotechnology and Applied Biochemistry, 1988, 10:143-153.
18. Campbell DH , Cherkin A . The destruction of pyrogen by hydrogen peroxides.Science,1945,102:535-538.
19. Liu Shi-gu,Rowel Tobias,hannon Mcclure,et al.Removal of endotoxin from recombinant protein preparations . Clinical Biochemistry ,1997,30(6):455-463.
20. 商振华,周冬梅,郭为,等.去除内毒素的柱亲和介质与膜亲和介质的制备和特征.分析化学,1997,25(9):1010-1015.
21. 商振华,周冬梅,郭为,等.聚酰胺亲和膜色谱用于去除内毒素的研究.分析测试学报,1998,17(2):8-12.
22. 陈兆安,邓麦村,叶震,等.膜吸附去除内毒素的传质研究.大连理工大学生物医学工程学术论文集,2003Nov,1:364-36.7.
2. 李春梅,黄天卫,傅永潜.肝硬化患者内毒素测定的临床意义.实用内科学杂志,1988,8(1): 294-295.
3. 刘天舒,陈世耀,蔡鸿菊,等.自身腹水浓缩回输治疗难治性腹水的回顾性分析.中国临床医学,2003,10(2):164-166.
4. 朱宁川,陈岩.肝病肠道菌群失调与肠源性内毒素血症.中国微生态学杂志,2004,16(1): 61-62.
5. 周 喜 汉 . 肝 硬 化 顽 固 性 腹 水 治 疗 进 展 . 右 江 民 族 医 学 院 学报,2003,25(4):564-565.
6. 陈兆安.CA/PE 微孔亲和膜的制备及其性能与应用的研究.中国科学院大连化学物理研究所博士论文,2004.
7. A. Bruil, Leukocyte filters: Filtration mechanisms and material design. 1993,15-08.
8. D.Petsch, W.-D.Dechwer, F.B.Anspach, et al. Endotoxin removal with poly(ethyleneimine)-immobilized adsorbers: Sepharose 4B versus flat sheet and hollow fibre membranes. J.Chtomatogr.B,1998,707:121-130.
9. D.Petsch,T.C. Beeskow, F.B.Anspach, et al .Membrane adsorbers for selective removal of bacterisl endotoxin.J.Chtogr.B, 1997,693:79-91.
10. 中华人民共和国药典.2005 年版,二部,化学工业出版社.
11. 王思理主编.细菌内毒素检查法-定量法.
12. 魏桂林.亲和膜用于医药及体液中内毒素等的去除研究.中国科学院大连化学物理研究所硕士论文,2000.
13 D.Petsch,F.B.Anspach, Endotoxin removal from protein solutions.J. Biotech,2000,76:97-119.
14. Y.Kang,RG.Luo.Effects of ionic strength and PH on endotoxin removal Efficxiency and protein recovery in an affinity chromatography. Proc.Biochem,2000,36:85-92.
15. 商振华,周冬梅,郭为,等.聚酰胺亲和膜用于去除内毒素的研究.分析测试学报,1998,17(2):8-10.
16. 魏桂林,商振华,于亿年,等.新型亲和膜色谱用于去除胆红素的研究.色谱,2001,19(1):74-77.
1. 罗海波,朱平,李兰娟,等编.细菌毒素与临床[M].北京,人民卫生出版社,1999,9-10.
2. 赵龙凤,韩德武.肝病患者内毒素血症的临床意义.世界华人消化杂志,1999.7(5):391.
3. 姚咏明,张立天,陆家齐,等.脓毒症大鼠内毒素增敏系统改变与高迁移率族蛋白-1 表达的关系.中华创伤杂志,2002,18(9):540-543.
4. 唐虹.内毒素及内毒素血症治疗研究进展.医学综述,2005,11(2):109-110.
5. 中村东树他.肝病洞壁细胞研究の进展.东京,国际医书出版,1990,248.
6. Fox ES, Thomas,Broitman SA. The macrophage 80 kda endotoxin binding protein is not present on rat kupffer cells . Gastroenterology, 1990,98:586.
7. Shakamoto W,Nishihiral, Fujie K, et al.[J].Nutr Sci Vitaminol(Tokyo),2000Dec, 46(6):316.
8. 王顺宾.抗生素诱导革兰阴性杆菌释放内毒素的研究进展.国外医药抗生素分册,1998, 19(5):342-343.
9. Bucklin SE,Lake P, Logdberg L, et al. Therapeutic efficacy of a polymyxin B-dextran 70 conjugate in experimental model of endotoxemia. Antimicro Agens chemother.1995, 39(7):1462-1466.
10. 陆付耳,李鸣真,叶望云,等.话内毒素血症的治疗对策.中国危重病危急救医学,2000, 12(10):579.
11. 梅 受 敏 , 张 淑 慧 . 治 疗 肝 病 内 毒 素 血 症 进 展 . 中 国 医 院 药 学 杂志,2003,23(2):111-112.
12. 冯俊明,史景泉,刘友生.脂多糖对枯否细胞中 CD14, Toll 样受体 4 表达的影响及其意义.中华实验外科杂志,2002,19(4):350-352.
13. 楼福乐,毛伟钢,陆晓峰,等.超滤技术在制药工业中除热原的应用.膜科学与技术,1999, 19(3):8-12.
14. 龚承元,苏建勇,朱孟府.高电微孔滤膜的制备及其性能的初步研究.中国药学杂志,1994, 29(4):214-217.
15. 苏建勇,龚承元,朱孟府.高电微孔滤膜去除细菌内毒素的试验研究.军事医学科学院院刊,1995,19(4):289-292.
16. Howard F,Spooner ECR.Removal of taints from water . chem Ind,1946,186-189.
17. Statoshi Minobe,Taizo Watanabe,Tadashi Sato,et al.Characteristics and applications of adsorbents for pyrogen remova.Biotechnology and Applied Biochemistry, 1988, 10:143-153.
18. Campbell DH , Cherkin A . The destruction of pyrogen by hydrogen peroxides.Science,1945,102:535-538.
19. Liu Shi-gu,Rowel Tobias,hannon Mcclure,et al.Removal of endotoxin from recombinant protein preparations . Clinical Biochemistry ,1997,30(6):455-463.
20. 商振华,周冬梅,郭为,等.去除内毒素的柱亲和介质与膜亲和介质的制备和特征.分析化学,1997,25(9):1010-1015.
21. 商振华,周冬梅,郭为,等.聚酰胺亲和膜色谱用于去除内毒素的研究.分析测试学报,1998,17(2):8-12.
22. 陈兆安,邓麦村,叶震,等.膜吸附去除内毒素的传质研究.大连理工大学生物医学工程学术论文集,2003Nov,1:364-36.7.