Fe～（3+）改性羧甲基纤维素预防术后腹膜粘连的作用及其机制的初探
摘要
腹部手术后腹腔粘连在普外科发病率高达90％，虽然现代外科手术的技术和方法已有很大发展，但仍未减少其发生率，研究表明，接受腹部中、小手术病人中1．2％及大手术病人中3．6％因术后肠粘连造成肠梗阻需要手术治疗，其中部分病人可能再次发生肠粘连，以致形成重度恶行循环。因此，预防术后腹腔粘连及粘连性肠梗阻一直是外科医师最为关注的课题之一。腹腔粘连的形成，缘于腹膜间皮细胞纤维蛋白原的释放和纤维蛋白溶解之间平衡关系的破坏，诸如机械性损伤、组织缺血、外源性物质的植入或腹膜的炎症等原因均可破坏这种平衡关系，使纤维蛋白原释放增加。同时，纤维蛋白溶解酶原激活物的活性降低，大量纤维蛋白沉积并机化形成永久的纤维性粘连，因此，最初预防术后腹腔粘连的基本措施主要是通过改进手术技术来减少机械性损伤；通过手术时避免大块结扎，保持组织良好的血液供应来防止组织缺血，通过药物来减少组织炎性反应。后来，研究人员又发现了许多预防腹腔粘连的辅助方法，主要有：用肝素来防止纤维蛋白的沉积；用重组组织型纤溶酶原激活剂来促进纤维蛋白的溶解；用己丁糖来抑制成纤维细胞的增生；用透明质酸酶增加乳化脂肪的吸收，用抑肽酶调节腹膜修复中的一些介质和纤维蛋白溶解作用等，但由于种种原因，以上方法均未能取得令人满意的效果。
进一步研究证实，病理状态下腹腔内两处或两处以上浆膜面的有效接触是形成粘连的必要条件，因此，人们尝试应用局部隔离物 质(包括膜状、液状物质)来预防腹部术后的腹腔粘连，多次的 实践及研究结果证明用来预防粘连的材料应具备以下特性：1、良好的生物相容性2、材料本身及其降解物无毒副作用，无诱变性3、在体内可完全降解且降解速度可人工控制。因此，合成可降解的生物高分子材料已成为生物材料研究和应用的热点。我们与中科院长春应用化学研究所联合研制了一种新的、可降解的医用生物高分子材料：羧甲基纤维素及金属离子Fe3+改性羧甲基纤维素，后者除具以上特性外，其在体内降解时间与病理肠粘连时间基本相同，其确切的抗粘连效果已经得到证实。其抗粘连的最佳浓度为3％，进一步得出最佳剂量为45mg/Kg 体重。
在上述工作的基础上，我们设计并进行了本实验：目的 研究Fe3+-CMC预防术后腹膜粘连的机制。方法 1、术后24小时取实验动物血清，测定tPA、uPA含量变化，评价Fe3+-CMC抑制术后炎性反应情况；2、于术后1天、3天、5天、7天、14天、2月时，通过大体观察，组织学观察，评价Fe3+-CMC预防术后腹膜粘连的情况及粘连部位的组织学改变；3、应用免疫组织化学技术（SABC），测定粘连组织中TGF-β,FGF的表达，从而观察Fe3+-CMC对术后腹膜损伤部位TGF-β,FGF
表达的抑制作用；4、通过透射电镜观察粘连组织中炎细胞功能状态的变化。结果 1、术后24小时实验组动物血清中tPA、uPA含量明显高于对照组（p〈0.01〉；2、实验组动物术后腹膜粘连明显减轻，分级均在0～Ⅱ级，对照组粘连分级均在Ⅲ～Ⅳ级；组织学观察可见实验组粘连组织胶原纤维沉积、炎细胞反应明显低于对照组；3、实验组动物粘连组织中TGF-β、FGF表达明显低于对照组（p<0.01），说明Fe3+-CMC有明显抑制TGF-β、FGF表达的作用。4、透射电镜观察可见实验组动物粘连组织中巨噬细胞、成纤维细胞功能状态明显低于对照组。结论 Fe3+-CMC预防术后腹膜粘连的机制可能在于：1、Fe3+-CMC起到了良好的机械隔离作用；2、Fe3+-CMC能够抑制炎性反应，提高血清及损伤局部组织中的tPA,uPA的水平；3、Fe3+-CMC能够减少腹膜损伤局部的炎性细胞的渗出，减少胶原蛋白沉积，促进损伤腹膜的生理性完整修复；4、Fe3+-CMC能够抑制腹膜损伤后TGF-β的过量合成及表达；5、Fe3+-CMC抑制腹膜损伤后FGF的过量表达。
The incidence of the intestinal adhension after abdominal operation is as high as 90%, so the intestinal adhesion is still one of the more common complications although with advancement in science and improvement in surgical operation. One study has showed that 1.2-3.6% of the patients who receive abdominal operation will be reoperated just because of intestinal adhesion. For more severe case, mesenteriopexy in needed. This is a very big personal and social problem. Therefore, how to prevent iintestinal adhesion and obstruction after abdominal operation is one of the question which most surgeon pay attention to.
To prevent the postoperative peritoneal adhesion, we must understand the mechanism of the peritoneal adhesion firstly. Under normal conditions, the release Of fibrinogenase of the peritoneal mesothelium cells and fibrinolysis is kept balance, but the balance may be interrrupted by the mechanical incision, tissue ischemia, exogenous material implantation and peritonitis, which makes the release of fibrinogenase increased
and the activity of plasminogen activator decreased, resulting in lots of fibrin deposition and organization and formation of fibrous adhesion eventually. Therefore,the main measurement in prevention of the postoperative peritoneal adhesion is to reduce mechanical iincision by careful operation; to prevent tissue ischenmia by maintaining well blood supply; to diminish infammatory reaction by some medcine. The researcher had developed many supplementary measurements to prevent the postoperative peritoneal adhesion. For example, to prevent fibrin deposition by the heparin to promote fibrinolysis by rt-Pa, to restrain the proliferation of fibroblast by chitoscan, but the measurements are not very effective for some reasons.
It is verified that the effective contact of serious coat is necessary to form adhesion under the pathological state, many researchers had tried to utilize the llocal isolation materials (including membranous or liquid) to prevent the postoperative peritorial adhesion, but the methods can not be applied effectively, because the uncontrollable retain in the peritoneal cavity and the toxic and side-effect of the material
itself.
With the development of bioogical medcine, synthesis of biodegradable macromolecule material and it's application hasbecome one of the hot-spots, which provide the developing direction in using the local isolation material to prevent the peritoreal adhesion.The material of preventing postoperative peritoneal adhension should have the specific properties: 1. very well biocompatibility. 2. no toxic and side-effect. 3. completely degraded in the body with the Changchun Application Chemical Research Institute some kinds of the biodegradable macromolecule materials with the properties mentioned above. First, the intestinal adheaion model in rats was established on the basis of this, the experiment was performed with different kinds of macromolecule materials and found that the most
effective material is Fe3+ modify sodium carboxymethyl cellulose; the most effective density is 3%; the most ettective dose is45mg/Kg.
Based on the premior job, we continue to design and do the following experiments: Objective To study the mechanism of
Fe3+-CMCpreventingpostoperativeperitonealadhesion.Methods1.Measuring the variation of tPA,uPA to evaluate the effectiveness of Fe3+-CMC preventing postoperative inflammatory response;2.To ebasluate the efficacy Fe3+-CMC preventing postoperative adhesion by eye and microscopic oberservation, and observing the histologicalchangingofadhesivetissue.3.Measuring the expression ofTGF-β、FGF of adhesion tissue, then observing the effectiveness of Fe3+-CMC.4.Obersving the fundational varation of all kinds of cells in adhesive tissue by transmission electron microscopy.Results:1.The amount of (in the serum of experimental group is less than that of control group(p<0.01).2.The adhesion extent of experimental group is less than that of control group.it can be oberserved that the d
进一步研究证实，病理状态下腹腔内两处或两处以上浆膜面的有效接触是形成粘连的必要条件，因此，人们尝试应用局部隔离物 质(包括膜状、液状物质)来预防腹部术后的腹腔粘连，多次的 实践及研究结果证明用来预防粘连的材料应具备以下特性：1、良好的生物相容性2、材料本身及其降解物无毒副作用，无诱变性3、在体内可完全降解且降解速度可人工控制。因此，合成可降解的生物高分子材料已成为生物材料研究和应用的热点。我们与中科院长春应用化学研究所联合研制了一种新的、可降解的医用生物高分子材料：羧甲基纤维素及金属离子Fe3+改性羧甲基纤维素，后者除具以上特性外，其在体内降解时间与病理肠粘连时间基本相同，其确切的抗粘连效果已经得到证实。其抗粘连的最佳浓度为3％，进一步得出最佳剂量为45mg/Kg 体重。
在上述工作的基础上，我们设计并进行了本实验：目的 研究Fe3+-CMC预防术后腹膜粘连的机制。方法 1、术后24小时取实验动物血清，测定tPA、uPA含量变化，评价Fe3+-CMC抑制术后炎性反应情况；2、于术后1天、3天、5天、7天、14天、2月时，通过大体观察，组织学观察，评价Fe3+-CMC预防术后腹膜粘连的情况及粘连部位的组织学改变；3、应用免疫组织化学技术（SABC），测定粘连组织中TGF-β,FGF的表达，从而观察Fe3+-CMC对术后腹膜损伤部位TGF-β,FGF
表达的抑制作用；4、通过透射电镜观察粘连组织中炎细胞功能状态的变化。结果 1、术后24小时实验组动物血清中tPA、uPA含量明显高于对照组（p〈0.01〉；2、实验组动物术后腹膜粘连明显减轻，分级均在0～Ⅱ级，对照组粘连分级均在Ⅲ～Ⅳ级；组织学观察可见实验组粘连组织胶原纤维沉积、炎细胞反应明显低于对照组；3、实验组动物粘连组织中TGF-β、FGF表达明显低于对照组（p<0.01），说明Fe3+-CMC有明显抑制TGF-β、FGF表达的作用。4、透射电镜观察可见实验组动物粘连组织中巨噬细胞、成纤维细胞功能状态明显低于对照组。结论 Fe3+-CMC预防术后腹膜粘连的机制可能在于：1、Fe3+-CMC起到了良好的机械隔离作用；2、Fe3+-CMC能够抑制炎性反应，提高血清及损伤局部组织中的tPA,uPA的水平；3、Fe3+-CMC能够减少腹膜损伤局部的炎性细胞的渗出，减少胶原蛋白沉积，促进损伤腹膜的生理性完整修复；4、Fe3+-CMC能够抑制腹膜损伤后TGF-β的过量合成及表达；5、Fe3+-CMC抑制腹膜损伤后FGF的过量表达。
The incidence of the intestinal adhension after abdominal operation is as high as 90%, so the intestinal adhesion is still one of the more common complications although with advancement in science and improvement in surgical operation. One study has showed that 1.2-3.6% of the patients who receive abdominal operation will be reoperated just because of intestinal adhesion. For more severe case, mesenteriopexy in needed. This is a very big personal and social problem. Therefore, how to prevent iintestinal adhesion and obstruction after abdominal operation is one of the question which most surgeon pay attention to.
To prevent the postoperative peritoneal adhesion, we must understand the mechanism of the peritoneal adhesion firstly. Under normal conditions, the release Of fibrinogenase of the peritoneal mesothelium cells and fibrinolysis is kept balance, but the balance may be interrrupted by the mechanical incision, tissue ischemia, exogenous material implantation and peritonitis, which makes the release of fibrinogenase increased
and the activity of plasminogen activator decreased, resulting in lots of fibrin deposition and organization and formation of fibrous adhesion eventually. Therefore,the main measurement in prevention of the postoperative peritoneal adhesion is to reduce mechanical iincision by careful operation; to prevent tissue ischenmia by maintaining well blood supply; to diminish infammatory reaction by some medcine. The researcher had developed many supplementary measurements to prevent the postoperative peritoneal adhesion. For example, to prevent fibrin deposition by the heparin to promote fibrinolysis by rt-Pa, to restrain the proliferation of fibroblast by chitoscan, but the measurements are not very effective for some reasons.
It is verified that the effective contact of serious coat is necessary to form adhesion under the pathological state, many researchers had tried to utilize the llocal isolation materials (including membranous or liquid) to prevent the postoperative peritorial adhesion, but the methods can not be applied effectively, because the uncontrollable retain in the peritoneal cavity and the toxic and side-effect of the material
itself.
With the development of bioogical medcine, synthesis of biodegradable macromolecule material and it's application hasbecome one of the hot-spots, which provide the developing direction in using the local isolation material to prevent the peritoreal adhesion.The material of preventing postoperative peritoneal adhension should have the specific properties: 1. very well biocompatibility. 2. no toxic and side-effect. 3. completely degraded in the body with the Changchun Application Chemical Research Institute some kinds of the biodegradable macromolecule materials with the properties mentioned above. First, the intestinal adheaion model in rats was established on the basis of this, the experiment was performed with different kinds of macromolecule materials and found that the most
effective material is Fe3+ modify sodium carboxymethyl cellulose; the most effective density is 3%; the most ettective dose is45mg/Kg.
Based on the premior job, we continue to design and do the following experiments: Objective To study the mechanism of
Fe3+-CMCpreventingpostoperativeperitonealadhesion.Methods1.Measuring the variation of tPA,uPA to evaluate the effectiveness of Fe3+-CMC preventing postoperative inflammatory response;2.To ebasluate the efficacy Fe3+-CMC preventing postoperative adhesion by eye and microscopic oberservation, and observing the histologicalchangingofadhesivetissue.3.Measuring the expression ofTGF-β、FGF of adhesion tissue, then observing the effectiveness of Fe3+-CMC.4.Obersving the fundational varation of all kinds of cells in adhesive tissue by transmission electron microscopy.Results:1.The amount of (in the serum of experimental group is less than that of control group(p<0.01).2.The adhesion extent of experimental group is less than that of control group.it can be oberserved that the d
引文
1.Haney AF.Prevention of postoperative abdominal adhesion by tissue precoating with polymer solution. Fertil Steril 1994,61(6):767
2.Scott-CoomberS, WhawellSA, VipondMN, et al. Human intraperitoneal fibrinolytic response to surgery [J].Br J Surg, 1995,82(3):414.
3.Pictleman J,Lee RM. The management of patients with suspected early postoperative small bowel obstruction. Am, J, Surg, 1998,210(2)126～219.
4.黄家驷主编，外科学，第四版。北京人民出版社；1986，1608～1069。
5.孙鸿斌、李有柱，腹部手术后肠粘连病因及预防。吉林医学；2000，20（3）：321。
6.SahiN, SkinnerK, ColtMJ. Reduction of Postoperative Pelvic adhesion. Acta obstet Gynicol Scand, 1994,73(1):70.
7.Lai HS, Chen Y. Effect of octrtide on postoperative intraperitoneal adhesion in rats. Sacand J Gastroenterol,1996,13(6):678.
8.Kathleen E, Rodger S. Reduction of adhesion formation by introperitonealadministrationofArg-Giy-AspcontainingpeptidesFertil,1998,709(6):1131.
9.WisemanD.Polymericsite-specificpharmacotherapy. Chichester:John
Wiley,1994,369.
10.Menzies D. Inhibitor of postsurgical adhesion in a standardized rabbit model.J R Soc Med, 1989,82:534.
11.Yusuf,OzogulL, TokaT, er al. An experimental study of the effect of aprotinin on intestinal adhesion formatoin, Am J Surg, 1998,175(2):137.
12.GalilliY, AvivT, Ben-AbrahamR, et al. Reduction of Surgery indrcePeritoneal Adhesions by Methylene Bluce,Am J Surg;1998,175(1):30～32.
13.Lucas PA, Warejcka DJ, Young HE, et al, Formation lf abdminal adhesions is inhibited by antibodies to transforming growth factor-betal. J Surg Res; 1996,65(2):135～138.
14.Holschneider CH. Immunodeviation with interlenkin-10 and intorleukin-4 compared with kerorolac tromethamine for prevention of postoperative adhesion in a murine model, J Surg Res;1997,70:138～143.
15.Andrew K, SaltzaN, OisonTH et al, Prevention of postoperative adhesions by an antibody to vascular permeability factor vascular endothelial growth factor in a murine model. Am J Obstet
Gynecol;1996,174(5);1502-1506.
16.顾其胜，医用透明质酸钠在临床的应用，中国修复重建外科杂志；1998，121（4）：314。
17.ArrajaB, Marko S, LarssonK, rt al, Exogenous phospholipid Reduces postoperative Peritoneal Adhesions in Rats Eur J Surg,1995,161(5):341.
18.Interceed Adhesion Barrier Study Group. J Surg Gynecol Obstet,1993,177:135.
19.曹文刚，腹膜粘连的发生及预防。中国实用外科杂志；2001，21（3）：170-172。
20.Ellis H. The cause and prevention of postoperioneal adhesion. Surg Gynecol Obsrtet,1971,133(3):479.
21.Fowler JM, Nisbet JD, Settles H, et al. Observasion on the pathogenesis of peritoneal adhesion. Gynecol Oncol, 1991,43(2):141.
22.Satish P> Local release lf fibrinolytic agents for adhesion prevention. J oj Biomat Applic, 1992,6(3):216.
23.武忠弼主编， 病理学（第四版）1994，9。
24.贺石林主编， 临床生理学， 1995，10。
25．Jorgensen JO, Lalok NJ,Huunt DR. Effect of octrtide on postoperative intraperitoneal adhesion in rats. Sacand J Gastroenterol,1996,13(6):678.
26.翁永强 转化生长因子-β多克隆抗体预防腹腔粘连的实验研究，中国普通外科杂志，2002，12，11（12）：741～745。
27.Chellai am, stucci AF, Cheugini N, et al. Role of transforming growth factor bata-1 in peritonitis-inducedadhesions[J].JGastrointestin Surg,2000,4(3):316～323.
28.Wayne AB, Et al. N Engl J Med,1994;331(9):1286～1291.
29.Milligan DM,Raftery AT.Observation on the pathogenesis of peritoneal adhesion.Br J Surg, 1974,61(4):274.
30.Ellis H. The aetiology lf post-operative abdominal adhesions.Br J Surg,1962,50(3):10.
31.LenaHolmdahl.PhD.Fibrinolysidinumanperitoneumduringoperation.Surgery,2000,119(6)701～705.
32.翁永强 β-生长因子与腹膜粘连， 国外医学，2000，27（2）：94～96。
33.Heinonen PK.Gynecol Obstet Invest 1990,29(4):292～295.
34.Thompson JN. Br J Surg 1989,76(4):382-384.
35.Vipind MN. Lancet 1990,335(1):1120～1126.
36.何有娣，黎燕 TGF-β和VEGF在血管生成中的作用 国外医学药学分册 2000，23（2）15～17。
2.Scott-CoomberS, WhawellSA, VipondMN, et al. Human intraperitoneal fibrinolytic response to surgery [J].Br J Surg, 1995,82(3):414.
3.Pictleman J,Lee RM. The management of patients with suspected early postoperative small bowel obstruction. Am, J, Surg, 1998,210(2)126～219.
4.黄家驷主编，外科学，第四版。北京人民出版社；1986，1608～1069。
5.孙鸿斌、李有柱，腹部手术后肠粘连病因及预防。吉林医学；2000，20（3）：321。
6.SahiN, SkinnerK, ColtMJ. Reduction of Postoperative Pelvic adhesion. Acta obstet Gynicol Scand, 1994,73(1):70.
7.Lai HS, Chen Y. Effect of octrtide on postoperative intraperitoneal adhesion in rats. Sacand J Gastroenterol,1996,13(6):678.
8.Kathleen E, Rodger S. Reduction of adhesion formation by introperitonealadministrationofArg-Giy-AspcontainingpeptidesFertil,1998,709(6):1131.
9.WisemanD.Polymericsite-specificpharmacotherapy. Chichester:John
Wiley,1994,369.
10.Menzies D. Inhibitor of postsurgical adhesion in a standardized rabbit model.J R Soc Med, 1989,82:534.
11.Yusuf,OzogulL, TokaT, er al. An experimental study of the effect of aprotinin on intestinal adhesion formatoin, Am J Surg, 1998,175(2):137.
12.GalilliY, AvivT, Ben-AbrahamR, et al. Reduction of Surgery indrcePeritoneal Adhesions by Methylene Bluce,Am J Surg;1998,175(1):30～32.
13.Lucas PA, Warejcka DJ, Young HE, et al, Formation lf abdminal adhesions is inhibited by antibodies to transforming growth factor-betal. J Surg Res; 1996,65(2):135～138.
14.Holschneider CH. Immunodeviation with interlenkin-10 and intorleukin-4 compared with kerorolac tromethamine for prevention of postoperative adhesion in a murine model, J Surg Res;1997,70:138～143.
15.Andrew K, SaltzaN, OisonTH et al, Prevention of postoperative adhesions by an antibody to vascular permeability factor vascular endothelial growth factor in a murine model. Am J Obstet
Gynecol;1996,174(5);1502-1506.
16.顾其胜，医用透明质酸钠在临床的应用，中国修复重建外科杂志；1998，121（4）：314。
17.ArrajaB, Marko S, LarssonK, rt al, Exogenous phospholipid Reduces postoperative Peritoneal Adhesions in Rats Eur J Surg,1995,161(5):341.
18.Interceed Adhesion Barrier Study Group. J Surg Gynecol Obstet,1993,177:135.
19.曹文刚，腹膜粘连的发生及预防。中国实用外科杂志；2001，21（3）：170-172。
20.Ellis H. The cause and prevention of postoperioneal adhesion. Surg Gynecol Obsrtet,1971,133(3):479.
21.Fowler JM, Nisbet JD, Settles H, et al. Observasion on the pathogenesis of peritoneal adhesion. Gynecol Oncol, 1991,43(2):141.
22.Satish P> Local release lf fibrinolytic agents for adhesion prevention. J oj Biomat Applic, 1992,6(3):216.
23.武忠弼主编， 病理学（第四版）1994，9。
24.贺石林主编， 临床生理学， 1995，10。
25．Jorgensen JO, Lalok NJ,Huunt DR. Effect of octrtide on postoperative intraperitoneal adhesion in rats. Sacand J Gastroenterol,1996,13(6):678.
26.翁永强 转化生长因子-β多克隆抗体预防腹腔粘连的实验研究，中国普通外科杂志，2002，12，11（12）：741～745。
27.Chellai am, stucci AF, Cheugini N, et al. Role of transforming growth factor bata-1 in peritonitis-inducedadhesions[J].JGastrointestin Surg,2000,4(3):316～323.
28.Wayne AB, Et al. N Engl J Med,1994;331(9):1286～1291.
29.Milligan DM,Raftery AT.Observation on the pathogenesis of peritoneal adhesion.Br J Surg, 1974,61(4):274.
30.Ellis H. The aetiology lf post-operative abdominal adhesions.Br J Surg,1962,50(3):10.
31.LenaHolmdahl.PhD.Fibrinolysidinumanperitoneumduringoperation.Surgery,2000,119(6)701～705.
32.翁永强 β-生长因子与腹膜粘连， 国外医学，2000，27（2）：94～96。
33.Heinonen PK.Gynecol Obstet Invest 1990,29(4):292～295.
34.Thompson JN. Br J Surg 1989,76(4):382-384.
35.Vipind MN. Lancet 1990,335(1):1120～1126.
36.何有娣，黎燕 TGF-β和VEGF在血管生成中的作用 国外医学药学分册 2000，23（2）15～17。