大鼠肾脱细胞支架的制备研究

Preparation of decellularized kidney scaffolds in rats

  • 摘要:
      背景  基于完整天然肾的脱细胞支架可以用于生物人工肾,然而目前对肾脱细胞支架的制备没有统一的标准。由于脱细胞的结果直接关系到再细胞化和肾功能的发挥,因此对脱细胞支架的制备方案进行探索和优化显得尤为重要。
      目的  探究基于大鼠完整肾的脱细胞支架制备方案,为组织工程肾的制备提供生物支架材料。
      方法  通过给离体肾灌注十二烷基硫酸钠(sodium dodecyl sulfate,SDS)溶液洗脱肾内的细胞成分制备大鼠肾脱细胞支架,病理染色及透射电镜观察组织结构及细胞外基质保留情况,超声和超声造影观察支架完整性和血管网络通畅性,DNA和SDS定量检测支架内的核酸和洗脱剂的残留,细胞毒性检测脱细胞支架的安全性。
      结果  脱细胞时间随着灌注流速增加而减少,高流速灌注洗脱肾造成鲍曼氏囊面积变大而导致鲍曼氏囊破裂,且有细胞核成分残留。低流速下制备的脱细胞支架结构完整、细胞成分洗脱干净。0.5 mL/min流速下液体环境中灌注洗脱制得的脱细胞支架结构完整,DNA和SDS残留符合标准,超声探查见脱细胞支架完整,造影检查见完整血管网络,支架无细胞毒性。
      结论  制备大鼠肾脱细胞支架的最佳灌注流速是0.5 mL/min,超声和超声造影检查可以作为评价肾脱细胞支架的手段。

     

    Abstract:
      Background  Decellularized scaffolds based on natural kidneys can be used in bioartificial kidneys. However, there is no standard for the preparation of kidney decellularized scaffolds. Since the result of decellularization is directly related to the subsequent recellularization and the performance of kidney function, it is necessary to explore and optimize the preparation of decellularized scaffolds.
      Objective  To explore the preparation of decellularized scaffolds based on intact rat kidneys and provide bio-scaffold materials for the preparation of tissue-engineered kidneys.
      Methods  Preparation of decellularized rat kidney scaffold was done by perfusing the isolated kidney with SDS solution to elute the cellular components in the kidney. The tissue structure and extracellular mechanism retention were observed by pathological staining and transmission electron microscopy. The integrity of the scaffold and the patency of the vascular network were observed by ultrasound and contrast-enhanced ultrasound. The nucleic acid in scaffold and residues of eluent were quantitatively detected by DNA and SDS, and cytotoxicity test were used to verify the safety of the prepared decellularized scaffold.
      Results  The decellularization time decreased with the increase of the perfusion flow rate. The high flow rate perfusion eluted the kidney, which caused the area of Bowman's capsule to become larger and then ruptured, with residual nuclear components. The decellularized scaffold prepared at low flow rate had a complete structure without cell components, and the scaffold had no cytotoxicity. The structure of decellularized scaffold perfused and eluted in a liquid environment at a flow rate of 0.5 mL/min were integrity, with DNA and SDS residues meeting the standards. The decellularized scaffold was intact by ultrasound exploration, and a integrity vascular network was visible on contrast examination.
      Conclusion  The optimal flow rate for preparing rat kidney decellularized scaffold is 0.5 mL/min. Ultrasound and contrast-enhanced ultrasound can be used as a pre-examination method for renal decellularized scaffold.

     

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