解剖学报

解剖学报 ›› 2017, Vol. 48 ›› Issue (4): 477-481.doi: 10.16098/j.issn.0529-1356.2017.04.019

• 技术方法 • 上一篇    下一篇

大鼠单叶肝去细胞生物支架的制备与鉴定

秦雨萌1 周涛2 张璐2 刘裕2 王新旺1 王志斌3 梅劲3 陈胜华1*   

  1. 1.南华大学解剖学教研室，湖南 衡阳 421001;2.温州医科大学第二临床医学院临床医学系，浙江 温州 325035;3.温州医科大学解剖学教研室，浙江 温州 325035
  • 收稿日期:2016-10-17 修回日期:2017-02-20 出版日期:2017-08-06 发布日期:2017-08-06
  • 通讯作者: 陈胜华 E-mail:281775877@qq.com
  • 基金资助:
    国家自然科学基金资助项目;蒸湘学者计划;研究生创新课题

Preparation and identification of decellularized scaffold of a single lobe of liver  in rat

QIN Yu-meng1 ZHOU Tao2 ZHANG Lu2 LIU Yu2 WANG Xin-wang1 WANG Zhi-bin3 MEI Jin3 CHEN Sheng-hua1*   

  1. 1. Department of Anatomy，University of South China，Hu’nan Hengyang 421001, China; 2. the Second Clinical Medical College，Wenzhou Medical University，Zhejiang Wenzhou 325035, China; 3. Department of Anatomy，Wenzhou Medical University，Zhejiang Wenzhou 325035, China
  • Received:2016-10-17 Revised:2017-02-20 Online:2017-08-06 Published:2017-08-06
  • Contact: CHEN Sheng-hua E-mail:281775877@qq.com

摘要：

目的 通过灌注法制备大鼠单叶肝去细胞生物支架，并对其进行鉴定。 方法 健康成年SD大鼠20只，随机分为去细胞组和正常对照组，每组10只。去细胞组经门静脉灌胃针插管，恒温37℃依次灌注肝素化PBS溶液，1%Triton X-100（pH 7.5～8.0）及PBS溶液。HE、Masson染色及扫描电子显微镜观察组织学及超微结构改变；免疫荧光结合4’,6-二脒基-2-苯基吲哚（DAPI)观察2组胶原蛋白Ⅳ和Ⅰ、层黏连蛋白和纤维连接蛋白观察细胞外基质的主要成分；DNA定性和定量分析组织中残留DNA浓度和片段长度；聚甲基丙烯酸甲酯铸型观察肝脏内血管分布情况。 结果 Triton X-100灌注4h左右即可制备单叶肝脏去细胞生物支架。在灌注过程中，肝内细胞和细胞碎片逐渐被清洗，最终变成半透明状。HE、Masson、免疫荧光染色及扫描电子显微镜显示，去细胞组肝生物支架较完整地保留了细胞外支架的成分，未见明显细胞及细胞核成分残留；去细胞组支架DNA残留量较正常对照组下降了97.32%，琼脂糖凝胶电泳未见明显的DNA条带。血管铸型标本显示，去细胞组血管分布与正常对照组相仿，其分支完整、清晰。 结论 运用Triton X-100灌注法所制备的大鼠单叶肝生物支架去细胞彻底，较完整地保留细胞外基质和血管网络结构，是一种简单易行且较为理想的制备实验用单叶肝生物支架的方法。

关键词: 去细胞, 肝, 细胞外基质, 组织工程, 免疫荧光, 大鼠

Abstract:

Objective To prepare a single lobe of liver decellularized extracellular matrix bio-derived scaffold and to perform preliminary identification. Methods Twenty adult SD rats were randomly divided into two groups decellularization and control groups. In decellularization group, intravenous catheters were inserted through the portal vein to establish channels for a single lobe of liver perfusion successively with heparinized PBS, 1%Triton X-100（pH 7.5-8.0）and PBS in 37℃. After decellularization, the scaffold and native liver were observed through genomic DNA contact analysis, scanning electron microscope, HE and Masson’s staining. Immunofluorescence staining and vascular cast were used to observe changes in extracellular matrix constitution. Results The single lobe of liver was decellularized in 4 hours. Its cells and debris were cleaned gradually in the perfusion process and became translucent eventually. HE, Masson’s, immunohistochemistry staining and scanning electron microscope showed a lot of collagen fibers but no visible cell nuclei remained after decellularization. Quantitative analysis of DNA content within the scaffold showed a 97.32% decrease compared to the native liver. The agarose gel electrophoresis showed no DNA bands associated with the decellularized scaffold. Cast specimen showed that portal vein was still intact and clear compared with the native liver. Conclusion The method of perfusion with Triton X-100 can effectively remove all cellular components, and retain the extracellular matrix and vascular network structure well. It is a convenient and ideal preparation method to decellularize a single lobe of liver scaffold with tissue engineering.

Key words: Decellularization, Liver, Extracellular matrix, Tissue engineering, Immunofluorescence, Rat