脂肪组织干细胞移植对大鼠部分结肠切除术后渗漏的减少
1Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine,Utrecht University, 2Department of Otorhinolaryngology,Erasmus MC University Medical Center, 3Department of Clinical Sciences and Public Health, Faculty of Veterinary Science,Mahidol University, 4Department of Surgery,Erasmus MC University Medical Center, 5Department of Orthopaedics,Erasmus MC University Medical Center, 6Hill’s Pet Nutrition Inc.
Summary
对大鼠模型中脂肪组织衍生干细胞 (ASC) 的制备和移植进行了未充分缝合的结直肠吻合。这项新的应用表明, ASC 片可以减少结直肠吻合口漏。
Abstract
吻合口瘘是结直肠手术后的灾难性并发症。虽然目前的防渗漏方法有不同的临床疗效水平, 但目前尚不完善的解决方案。干细胞治疗使用 ASC 表可以提供一个解决这个问题。陶瓷被认为是有希望的候选者, 促进组织愈合, 因为他们的营养和免疫调节性能。在这里, 我们提供的方法, 以生产高密度 ASC 表, 移植到结肠直肠吻合的大鼠模型, 以减少渗漏。陶瓷形成的细胞表在热反应的文化菜肴, 可以很容易地分离。在移植当天, 进行了5缝合结直肠吻合的部分结肠切除术。动物被立即移植与 1 ASC 表每鼠。ASC 床单自发地附着在没有任何胶水, 缝合, 或任何生物材料的吻合。动物群在术后3和7天被献祭。与移植动物相比, 控制动物的吻合口脓肿和渗漏率较高。在本模型中, 结肠吻合术后的 ASC 片移植成功, 并伴有较低的漏率。
Introduction
部分结肠切除与原发性吻合术是一个常见的手术, 可以做许多疾病, 如结肠直肠癌, 克罗恩病和憩1,2。结直肠吻合术后最可怕的并发症是吻合口漏3。虽然已经确定了与吻合口漏相关的几个危险因素, 但防止渗漏的解决方案仍然未知4,5。
脂肪组织衍生基质细胞 (陶瓷) 与抗炎和营养性质的6,7, 使这些细胞有望候选的再生疗法8。陶瓷促进组织愈合的有效性表现在各种组织, 如心肌, 皮肤, 食管9,10,11,12,13。然而, 很少有关于使用陶瓷促进肠道愈合的报道。陶瓷局部移植通过 ASC 包膜 biosutures 或浆膜注射陶瓷的实验性结直肠吻合术14的愈合或没有预防吻合口瘘, 尽管更有利的吻合口愈合15。
局部移植陶瓷悬浮或与生物材料结合可能与细胞保留不足或移植细胞分配不足11。细胞板技术16,17提供了一个创新的方法, ASC 交付18,19。因此, 在以前的研究中, 提出了一种新的方法, 其中陶瓷组织在细胞片可以应用于实验性结直肠吻合20。本研究表明, ASC 片移植成功地减少了大鼠部分结肠切除术后结直肠吻合口渗漏。本文报告了 ASC 板的制备和手术移植技术。
Protocol
皮下腹部脂肪组织是由人类捐赠者获得的医学伦理委员会 (#MEC 2014-092), 荷兰鹿特丹的伊拉斯谟 MC 大学医疗中心批准。所有动物实验均经动物实验伦理委员会批准, 荷兰鹿特丹伊拉大学医学中心 (133-14-01)。 1. 人类陶瓷隔离和文化 为1克脂肪组织准备3毫升的隔离培养基。混合低葡萄糖 Dulbecco 的改良鹰的培养基 (LG-DMEM) 与10克/升牛血清白蛋白 (BSA) 和1克/升胶原酶1型。 <l…
Representative Results
图 1显示了这项研究的流程图, 其中描述了 ASC 片培养和结肠切除吻合的过程。图 2显示了 asc 板的显微形貌和在分离期间和之后的 asc 片的宏观外观。图 3显示了 ASC 板剥离和移植的不同步骤。图 4显示了 ASC 表在吻合线上的存在, 并在术后3天内预防渗漏。 <p class="jove_content" fo:keep-toge…
Discussion
吻合口漏是结肠切除术后最严重的不良事件。目前还缺乏预防吻合口破裂和渗漏的最佳技术。对生物材料阵列的局部应用进行了研究, 结果25、26、27不同。细胞疗法的目的是通过组织置换或通过分泌分泌刺激局部愈合来促进组织修复。
在这个大鼠模型中, ASC 片移植技术上是成功的。临床和组织学评价表明, …
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢 S.E.R. Hovius 博士, M.A.M. 博士 Mureau 和整形外科部的所有外科医生, 以收集皮下脂肪组织。p. Sukho 得到荷兰研究金计划 (NFP-12/435) 的赠款支持, 在进行这项学习期间。Y.M. Bastiaansen-Jenniskens 由荷兰关节炎基金会 (LP11) 提供资助。
Materials
| LG DMEM | Gibco, Life technologies | 22320022 | ASC isolation and culture |
| Collagenase type I | Gibco, Life technologies | 17100-01 | ASC Isolation |
| Bovine Serum Albumin | Sigma-Aldrich | A9418 | ASC Isolation |
| Fetal bovine serum | Gibco, Life technologies | 10270-106 | FBS, ASC isolation and culture |
| 3% acetic acid with methylene blue | Stemcell Technologies | 7060 | ASC Isolation |
| Gentamicin | Gibco, Life technologies | 15750-037 | ASC isolation and culture |
| Ampothericin B | Gibco, Life technologies | 15290-018 | ASC isolation and culture |
| Shaker (Gallenkamp Environmental Shaker Model 10X 400) | Akribis Scientific | F240 | ASC Isolation |
| Centrifuge | Hettichlab | Rotina380 | ASC isolation and culture |
| Phosphate buffer saline | Gibco, Life technologies | 14190-094 | PBS, ASC isolation and culture |
| Ascorbic acid-2-phosphate | Sigma-Aldrich | A8960 | ASC isolation and culture |
| Human recombinant fibroblast growth factor 2 | AbD Serotec | AF-100-15 | FGF2, ASC isolation and culture |
| Trypsin EDTA | Gibco, Life technologies | 25200-056 | ASC culture |
| Dimethyl sulfoxide | Sigma-Aldrich | D2650-5x | DMSO, ASC freezing |
| Thermo-responsive culture dishes | Nunc Thermo scientific | 174904 | ASC sheet preperation |
| Non-absorbable braided silk 4/0 | B Braun | 21151042 | surgery |
| Non-absorbable monofilament polyamide 8/0 | B Braun | G1118170 | surgery |
| Absorbable braided polyglycolic acid 5/0 | B Braun | C1048207 | surgery |
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Cite This Article
Sukho, P., Boersema, G. S., Kops, N., Lange, J. F., Kirpensteijn, J., Hesselink, J. W., Bastiaansen-Jenniskens, Y. M., Verseijden, F. Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model. J. Vis. Exp. (138), e57213, doi:10.3791/57213 (2018).