中国组织工程研究

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (2): 248-253.doi: 10.3969/j.issn.2095-4344.0014

• 材料生物相容性 material biocompatibility • 上一篇    下一篇

两种去污剂在制备脱细胞肺支架中的对比

马金辉，于  洁，乔叶薷，候陈玮，鞠志海，黑飞龙
  

  1. 北京协和医学院，中国医学科学院阜外医院，国家心血管中心，心血管疾病国家重点实验室，北京市 102308
  • 收稿日期:2017-08-28 出版日期:2018-01-18 发布日期:2018-01-18
  • 通讯作者: 黑飞龙，教授，主任医师，博士生导师，中国医学科学院阜外医院体外循环中心，北京市 102308
  • 作者简介:马金辉，男，1985年生，河南省郑州市人，汉族，北京协和医学院在读博士，主要从事组织工程人工肺及组织工程心脏瓣膜研究。
  • 基金资助:
    国家自然科学基金(31370993)

Comparative assessment of two detergents for decellularized lung scaffolds 

Ma Jin-hui, Yu Jie, Qiao Ye-ru, Hou Chen-wei, Ju Zhi-hai, Hei Fei-long
  

  1. State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, Chinese Academy of Medical Sciences & Beijing Union Medical College, Beijing 102308, China
  • Received:2017-08-28 Online:2018-01-18 Published:2018-01-18
  • Contact: Hei Fei-long, Professor, Chief physician, Doctoral supervisor, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, Chinese Academy of Medical Sciences & Beijing Union Medical College, Beijing 102308, China
  • About author:Ma Jin-hui, Studying for doctorate, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, Chinese Academy of Medical Sciences & Beijing Union Medical College, Beijing 102308, China
  • Supported by:
    the National Natural Science Foundation of China, No. 31370993

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摘要：

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文题释义：
组织工程肺：首先获取组织工程肺支架，通过种植宿主细胞，在生物反应器中进行细胞培养，最后移植到宿主体内，形成一个具有气体交换功能的人工肺，应用于临床。理论上讲，这种利用组织工程技术构建的人工肺解决了供体来源不足的问题，为肺移植提供了新的方向。
脱细胞肺支架：采用去污剂等去除了肺内的细胞成分，同时保留细胞外基质成分，维持了支架的三维结构，有利于促进宿主细胞的黏附、增殖、分化，移植体内后不需要应用任何免疫抑制剂，是一种理想的组织工程支架。
 
背景：制备脱细胞肺支架的一个困难在于必须充分去除细胞同时尽可能保留细胞外基质。灌注去污剂-核酸酶法已被广泛应用于脱细胞支架的制备。
目的：对比脱氧胆酸钠和十二烷基硫酸钠两种去污剂用于制备脱细胞肺支架的效果。
方法：取出24只雄性SD大鼠肺脏，分3组，正常对照组不进行任何干预；脱氧胆酸钠组灌注去污剂脱氧胆酸钠联合核酸酶，制备脱细胞肺支架；十二烷基硫酸钠组灌注去污剂十二烷基硫酸钠联合核酸酶，制备脱细胞肺支架；取3组肺组织，进行组织学染色、免疫荧光染色及DNA含量测定。将A549细胞分别接种于两组脱细胞肺支架上培养7 d，进行苏木精-伊红染色。将两组脱细胞肺支架分别埋于SD大鼠背部皮下，2周后进行移植组织Masson 染色。
结果与结论：①正常对照组肺组织布满细胞，两脱细胞肺支架均没有残留的细胞和细胞核，但脱氧胆酸钠组肺支架内肺泡形态结构较十二烷基硫酸钠组完整；两组肺支架的DNA含量均低于正常对照组(P < 0.01)；②培养7 d，两组肺支架上的细胞均是由边缘向组织中间生长、浸润，脱氧胆酸钠组肺支架内的细胞生长、浸润速度明显优于十二烷基硫酸钠组；③肺支架移植2周后，十二烷基硫酸钠组肺支架与周围组织融合欠佳，边界清楚，可见大量细胞浸润，分布较均匀，血管内血细胞清晰可见；脱氧胆酸钠组肺支架内新生血管数量明显多于十二烷基硫酸钠支架，并且新生血管直径更大；④结果表明与十二烷基硫酸钠相比，采用去污剂脱氧胆酸钠制备的脱细胞肺支架生物相容性更好，与周围组织融合速度更快，细胞浸润和新生血管形成更明显。

关键词: 生物材料, 组织工程, 脱细胞, 支架, 细胞外基质, 去污剂, 灌注, 黏附性, 细胞化, 血管化, 生物相容性, 国家自然科学基金

Abstract:

BACKGROUND: It is quite difficult to produce a decellularized lung scaffold, in which cells are removed and the extracellular matrix components (ECM) are preserved effectively. Perfusion of detergent-enzymes is an effective method with wide applications for decellularized lung scaffolds.
OBJECTIVE: To investigate the effects of two detergents (sodium deoxycholate, SDC and sodium dodecyl sulfate, SDS) on the preparation of decellularized lung scaffolds.
METHODS: Twenty-four male Sprague-Dawley rats were randomized into three groups: control group with no intervention, SDC group and SDS group. Decellularized lung scaffolds were prepared by perfusion of SDC or SDS combined with enzymes. The rat lung tissues in the three groups were taken for histological staining, immunofluorescent staining and DNA quantification. A549 cells were cultured and seeded onto the decellularized lung scaffolds for 7 days followed by hematoxylin-eosin staining. The decellularized lung scaffolds prepared by perfusion of SDC or SDS were subcutaneously implanted into the rat back, and the implants were retrieved and assessed by Masson staining after 2 weeks.
RESULTS AND CONCLUSION: In the control group, there were abundant cells in the lung tissues. In the other two groups, the decellularized lung scaffolds were nearly transparent, and the morphology of the SDC scaffold was more close to the native lung. There were no residual cells and nuclei on the two scaffolds, and the DNA content in the SDS and SDC groups was significantly lower than that in the control group (P < 0.01). At 7 days of culture, A549 cells cultured on the SDS and SDC scaffolds migrated from the edge to the center of the scaffold. Comparatively speaking, the migration ability of A549 cells on the SDC scaffolds was stronger, and there was obvious cell invasion and growth in the middle part of the lung. After 2 weeks of scaffold transplantation, the SDC implants poorly fused with the surrounding tissues, with a clear boundary, a large number of infiltrating cells distributed evenly, and intravascular blood cells were clearly visible; the number of new blood vessels with larger diameter in the SDC scaffold was significantly higher than that in the SDS scaffold. These findings indicate that the SDC scaffold has better biocompatibility than the SDS scaffold, which can fuse with the surrounding tissues faster and produce more infiltrating cells and new blood vessels.  

Key words: Lung, Extracellular Matrix, Tissue Engineering

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