靶向间皮角质细胞生长因子预防大鼠术后腹膜粘连的实验研究

目的 研究角质细胞生长因子(keratinocyte growth factor,KGF)通过促进间皮再生及影响腹膜纤溶活性而对大鼠腹膜粘连形成的预防作用.方法 30只雌性SD大鼠,平均分为3组,KGF组、阳性对照组和阴性对照组,每组10只.各组于术后第7天处死大鼠,参考Leach评分系统评估腹膜粘连程度,并采用免疫组化法来测定盲肠粘连组织中tPA、PAI-1表达情况.采用HE染色光镜观察组织变化情况,苦味酸天狼猩红染色-偏振光观察Ⅰ、Ⅲ型胶原蛋白表达.结果 KGF组胶原纤维变少,KGF组粘连总评分为(4.8±1.0)低于阳性对照组(7.6±1.0),两者差异有统计学意义(t=5.422,P＜0.01);KGF组Ⅰ型胶原纤维平均灰度值(69±11)明显高于阳性对照组(55±9)(t=3.214,P＜0.01);KGF组Ⅲ型胶原纤维平均灰度值(48±7)与阳性对照组(50±10)相比,差异无统计学意义(t=0.481,P＞0.05).免疫组化结果 示KGF组tPA表达水平(88±4.0)明显高于阳性对照组(112±4.0)和阴性对照组(101±2.0)(F=109.1,P＜0.01),PAI-1表达水平在3组之间的差异无统计学意义(F=1.391,P＞0.05).结论 KGF可促进腹膜间皮修复和增加间皮纤溶能力,抑制胶原沉积,从而降低术后粘连强度.

Abstract：

Objective To investigate the effects of keratinocyte growth factor on peritoneal adhesion formation in rats. Methods Thirty Sprague-Dawley( SD) female rats were randomly assigned to 3 groups, KGF group( n = 10), positive control group (n = 10) , and negative control group (n = 10). Seven days after surgery, rats were killed and the adhesion degree was evaluated by Leach scale. Immunohistochemical technique was used to identify the expression of tPA and PAI-1. Stained with HE,the histomorphology changes of the adhesion tissue were observed by light microscope. Picrosirius-polarization method was used to observe the expression of type Ⅰ or Ⅲ collagens in two groups. Results In the KGF group,lower collagen fibers were noted and the gross adhesion scores was significantly lower than that in positive control group (4. 8 ± 1. 0 vs 7. 6 ± 1. 0; t = 5.422; P ＜ 0. 01). The expression level of type Ⅰ collagens was significantly lower in the KGF group than in positive control group (69 ±11 versus 55 ±9;t = 3. 214 ;P ＜0. 01) ,but there was no significant difference in the expression of type Ⅲ collagens among the two groups (48 ± 7 versus 50 ± 10; t = 0. 481; P ＞ 0. 05). The immunohistochemistry showed that the expression of tPA significantly increased in the KGF group than in positive control group and negative control group(88 ±4.0 versus 112 ±4.0, 101 ±2.0;F = 109. l,P＜0. 01) , However, no statistically significant difference for the expression of PAI-1 was noted among the three groups ( F = 1. 391, P ＞ 0. 05). Conclusions Keratinocyte growth factor promotes mesothelium repair, increases mesothelial fibrinolytic activity, inhibits the deposition of collagen and reduces the intensity of postoperative adhesions.     

Phosphatidylcholine synthesis by peritoneal mesothelium: its implications for peritoneal dialysis

This study investigated the possibility that the peritoneum is capable of synthesizing phosphatidylcholine (PC), a lubricant surfactant, in an amount similar to that produced by pulmonary alveoli. The synthesis of PC by rat lung (positive control), liver (negative control), and transparent mesentery (test tissue) was determined by in vitro incubation of these tissues in the presence of (methyl-14C) choline chloride for three hours at 37 degrees C in Warburg flasks. All lipid material present in tissue and incubation media was extracted by the Folch technique. Carrier egg PC was added to each sample and total PC was isolated using high performance thin-layer chromatography. The PC fractions were counted for total radioactivity. No statistically significant difference was observed between the mean radioactive value for mesenteric tissue compared with lung tissue. The mean radioactive value for liver when compared with mesenteric and lung tissue was significantly lower (P less than 0.001). Thus, under the conditions of the experiment, we have demonstrated for the first time that peritoneal tissue is capable of synthesizing PC in amounts similar to that produced by the lung. Electron microscopy of transparent mesentery (test tissue) showed that mesothelium constituted the bulk of the cell population. Therefore, it is most likely that the PC that has been detected in peritoneal fluid is produced by the mesothelial cell. These findings are of significant relevance to developing concepts on the role of surfactant phospholipids in the physiology of peritoneal dialysis.     

Fecal peritonitis: Microbial adherence to serosal mesothelium and resistance to peritoneal lavage

Fecal contamination of the peritoneal cavity is a serious and potentially life-threatening event. While numerous models have been developed to study the pathogenesis of intraabdominal infection, to date, most investigations have failed to focus on the adherence of the contaminants to the serosal mesothelium. In the present investigation, the cecal ligation and puncture technique (CLP) was performed in Sprague-Dawley rats to study the following: (a) the kinetics of microbial adherence to the serosal mesothelium, (b) the stability of the aerobic and anaerobic intraperitoneal/mesothelial populations, following extended saline lavage, and (c) the impact of antimicrobial lavage on the stability of the mesothelial microbial populations. The Enterobacteriaceae rapidly colonized the serosal mesothelium and were the predominant flora up to 4 hours post-CLP. After 8 hours, the Bacteroides fragilis group represented the predominant peritoneal wash and mesothelial-associated microorganisms. Extended saline lavage failed to significantly reduce the mesothelial microbial populations. While antimicrobial lavage produced an immediate decrease in mesothelial microbial recovery, the results were transitory and the microbial populations achieved or exceeded prelavage levels at 24 hours postlavage. Microbial colonization of the peritoneal mesothelial surface is a rapid and stable phenomena following penetrating injury to the distal bowel. The results further suggest that the mesothelial populations are resistant to intraperitoneal lavage.

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Resumen La contaminación de la cavidad peritoneal representa un evento grave y potencialmente letal. Aunque se han desarrollado diversos modelos para estudiar la patogenesis de la infección abdominal, hasta la fecha la mayoría de los investigadores ha desatendido el estudio de la adherencia de los agentes contaminantes al mesotelio de la serosa. En la presente investigación se practicó la ténica de la ligadura y punción del ciego (LPC) en ratas Sprague-Dawley y con miras a estudiar los siguientes aspectos: (a) la cinética de la adherencia microbiana al mesotelio seroso, (b) la estabilidad de las poblaciones microbianas aeróbicas y anaerobicas intraperitoneales/mesoteliales después de un profuso lavado con solución salina, y (c) el impacto del lavado antimicrobiano sobre la estabilidad de las poblaciones microbianas mesoteliales. Las enterobacteriáceas colonizaron rápidamente el mesotelio seroso y fueron la flora predominante hasta 4 horas post-LPC. Después de 8 horas el grupo de losBacteroides fragilis representó los microorganismos predominantes en el lavado peritoneal y asociados con el mesotelio. El lavado profuso con solución salina no logró reducir en forma significativa las poblaciones microbianas mesoteliales. Aunque el lavado antimicrobiano produjo una reducción inmediata en la recuperación de microbios mesoteliales, los resultados fueron transitorios y las poblaciones microbianas lograron o excedieron los niveles prelavado a las 24 horas postlavado. La colonización microbiana de la superficie peritoneal mesotelial es un fenómeno rápido y estable después de una lesion penetrante del intestino distal. Los resultados también sugieren que las poblaciones microbianas mesoteliales son resistentes al lavado intraperitoneal.

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Résumé La contamination de la cavité péritonéale par des matières fécales est un événement grave qui peut menacer le pronostic vital. On a developpé de nobreux modèles expérimentaux pour étudier la pathogenèse de l'infection intra-abdominale mais à ce jour la plupart des études n'ont pas tenu compte de l'adhérence du contaminant de la séreuse. Dans cette étude, on a effectué la technique de ligature cécale et ponction (LCP) chez le rat Sprague-Dawley pour étudier: (a) la cintétique de l'adhérence microbienne à la séreuse, (b) la stabilité des populations aérobiques et anaérobiques intrapéritonéales et séreuses après lavage péritonéal, et (c) les conséquences du lavage antimicrobien sur la stabilité des populations microbiennes mésothéliales. Les Entérobactericeae colonisent rapidement le mésothélium et prédominent dans les 4 heures suivant la LCP. Après 8 heures, le germe le plus fréquent dans la cavité péritonéale et sur la séreuse est leBacteroïdis fragilis. Le lavage au sérum physiologique abondant n'a pas réduit de façon significative les populations microbiennes mésothéliales. Si le lavage à visée antibactérienne diminue la quantité de germes mésothéliaux récupérés, ce résultat est transitoire et les populations microbiennes atteignent ou dépassent le niveau du prélavage 24 heures après la lavage. La colonisation microbienne de la surface mésothéliale péritonéale est un phénomène rapide et stable après une plaie pénétrante de l'intestin distal. Ces résultats font supposer que les populations bactériennes mésothéliales résistent au lavage péritonéal.

     

Regeneration of peritoneal mesothelium in a rat model of peritoneal fibrosis

BACKGROUND: Patients on long-term peritoneal dialysis develop progressive peritoneal fibrosis and loss of mesothelial layer. Regeneration of the mesothelium has been reported in the normal peritoneum but not the fibrotic peritoneum. Moreover, the origin of the regenerated mesothelial cells remains obscure. The aim of this study was to investigate mesothelial regeneration in fibrotic peritoneum induced by chlorhexidine gluconate. METHODS: Peritoneal fibrosis was induced by injection of CG into the peritoneal cavity of Wistar rats. After injection, the abdomen was opened, and the parietal fibrotic peritoneum with mesothelial cells was stripped from the abdominal wall, and then the abdominal incision was closed. Rats were sacrificed, and peritoneal tissues were dissected out at 0, 1, 3, 5, or 7 days after the stripping procedure. RESULTS: Spindle-shaped cells with microvilli appeared on the surface of stripped peritoneum at day 3 after denudation. Immunohistochemistry identified staining for vimentin, a marker of mesoderm cells, in the spindle-shaped cells at days 3, 5, and 7. Expression of alpha-SMA was observed in the same cells at days 3 and 5, but not 7. Expression of cytokeratin and HBME-1, markers for mesothelial cells, in these cells was delayed until day 7. CONCLUSIONS: Mesothelium can regenerate on the fibrotic peritoneum. The regenerated mesothelial cells seem to originate from vimentin-positive mesenchymal cells.     

Laparoscopic treatment of peritoneal adhesions

Based on experience of treatment of 3000 patients with peritoneal comissures, new methods of diagnosis, prevention and treatment of this disease were developed and used. Exact verification of location and degree of comissures permits carrying out minimally invasive surgical procedures. A new variant of membranous allo-abdominal graft with anti-infective and anti-adhesive properties was developed and used for prevention of comissures. This approach leads to reduction of the number of ...     

Autologous implant of peritoneal mesothelium in rabbits and man

With the purpose of studying peritoneal physiology, original biopsy methods were devised and human and rabbit peritoneal mesothelial cells cultured and characterized. It was then decided to verify whether these cells could be implanted autologously during peritoneal dialysis in cases of acute loss of mesothelial surface. Staphylococcal peritonitis was induced in 12 rabbits and after 4 days of antibiotics, 6 of them were autoimplanted with cultured mesothelial cells, previously marked in 3 cases with thymidine (H3TdR). Implanted rabbits sacrificed after 3 and 6 days showed taking of the new mesothelial cells both by direct morphological observation and by autoradiography. Four uremic CAPD patients recovering from severe peritonitis were implanted with 300 million of their own peritoneal mesothelial cells, previously cultured and frozen. Morphological signs of taking were evident by laparoscopy and from peritoneal biopsies performed 3 and 6 days after implant. The fact that such implants are possible may have interesting applications in medicine and surgery.     

腹膜粘连无创评估手段的进展

腹膜粘连是指手术、创伤、腹腔感染、腹膜透析等病因导致的腹腔脏器和组织修复过程中形成的异常的纤维连接。腹部手术是腹膜粘连的最常见原因,绝大多数腹部手术患者术后均会出现不同程度的腹膜粘连。腹膜粘连可以导致患者出现长期的腹痛、诱发肠梗阻,部分患者可能因为粘连带的卡压而出现急性肠坏死。而在女性中,腹膜粘连增加了女性不孕的风险。除此之外,腹膜粘连可以使二次手术变得困难,术中容易出现副损伤。通过无创手段来评估腹膜粘连的程度,筛查腹膜粘连的高危人群,评估药物和屏障材料的临床效果,具有重要的临床意义。超声检查、磁共振电影成像和人工气腹CT能够对腹膜粘连进行精准评估。除此之外,体质量指数、炎症标志物以及赖氨酰氧化酶样蛋白2等指标也对粘连的评估有一定的价值。笔者对目前的腹膜粘连无创评估手段进行综述,以期为临床提供参考。     

Laparoscopic treatment of postoperative peritoneal adhesions

75 patients with postoperative peritoneal adhesions, complicated by a severe pain syndrome, were operated on during 2003-2006 yy. The preoperative use of the enzyme medication in 28 patients allowed therefore the conduction of adequate laparoscopic adhesiolysis. There were no complications and recurrences registered at follow-up. The postoperative hospital stay averaged 3.7 days.     

Seprafilm reduces adhesions to polypropylene mesh and increases peritoneal hydroxyproline

Polypropylene mesh is an effective and widely used material in repairing abdominal wall defects, but it causes dense adhesions when in contact with abdominal viscera directly. As a consequence of this process intestinal obstruction and enterocutaneous fistula may develop. The purpose of the present study was to determine whether Seprafilm, a bioresorbable translucent membrane, reduces abdominal visceral adhesions to polypropylene mesh and whether Seprafilm has any effects on peritoneal tissue hydroxyproline levels. Twenty-six adult Wistar rats were used. A full-thickness abdominal wall defect was created and cecal abrasions were created to induce adhesion formation in each animal. All of the abdominal defects were repaired with polypropylene mesh. In addition a Seprafilm membrane was laid over the abdominal viscera in the Seprafilm group (n = 13). The abdominal cavity was evaluated for adhesion formation, and peritoneal biopsies were taken for the measurement of tissue hydroxyproline levels at the 14th day. The use of Seprafilm resulted in significant reduction in the adhesion formation (P = 0.002) and a significant increase in peritoneal hydroxyproline level (P < 0.0001). These findings demonstrate that the increase of peritoneal hydroxyproline levels caused by Seprafilm might play a role on the antiadhesive effects of Seprafilm.