Institute of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, China.
Department of Cardiothoracic Surgery Lab, Changhai Hospital, Second Military Medical University, Shanghai, China.
Xenotransplantation. 2018 Mar;25(2):e12380. doi: 10.1111/xen.12380. Epub 2018 Feb 15.
Various detergent-based protocols are used to remove cells and cellular debris in porcine aortic valve (PAV). However, the removal of antigenic cellular components has not been thoroughly elucidated to date. In this study, we used 4 detergent-based protocols to decellularize PAVs and aimed to evaluate their effects on removing antigenic cellular components.
Porcine aortic valves were decellularized using sodium dodecyl sulfate (SDS), SDS in combination with sodium deoxycholate (SDS/SD), Triton X-100, and Triton X-100 in combination with SD (Triton X-100/SD), respectively. Untreated PAVs were used as controls. Immunohistochemical and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analyses were performed to determine the removal of antigenic protein components. Histological, biochemical, and biomechanical analyses were performed to determine the preservation and mechanical properties of the extracellular matrix. PAV tissues were implanted subcutaneously in Sprague Dawley rats to evaluate the host immune response. Implanted PAVs were taken out at the indicated time points for histological and immunohistochemical examinations.
All 4 protocols effectively removed the membrane antigenic proteins major histocompatibility complex I molecule and galactose-α-1,3 galactose. The SDS/SD protocol was the most effective method to remove the cytoplasmic cytoskeletal proteins, vimentin and α-SMA, and SDS alone partly removed vimentin protein. The SDS/SD protocol was the most effective method to remove nuclear DNA with residual DNA below 50 ng/mg, followed by the SDS protocol with residual DNA of 74.9 ng/mg. The SDS protocol was the most effective method to remove proteins ranged from 10 to 55 kDa, followed by the SDS/SD protocol. SDS and SDS/SD PAV implants attracted fewer neutrophils in vivo on postoperative day 3. The infiltration of macrophages and T lymphocytes was significantly lower in SDS and SDS/SD implants on days 14 and day 28. All of the decellularized protocols that were examined greatly reduced the contents of collagen, elastin, and glycosaminoglycan compared to controls. Biomechanical analysis revealed significant differences in ultimate tensile strength and Young's modulus between control PAVs and decellularized PAVs generated using SDS, SDS/SD, Triton X-100, or Triton X-100/SD.
These results indicated that SDS-based protocols more effectively removed antigenic cellular components compared to Triton X-100-based protocols. These results are clinically significant because complete removal of antigenic determinants is critical to decrease adverse immune-mediated and inflammatory responses to a PAV when used in xenogeneic application.
目前，人们采用了各种基于清洁剂的方法来去除猪主动脉瓣（PAV）中的细胞和细胞碎片。然而，到目前为止，对于去除抗原性细胞成分的方法还没有进行彻底的阐述。在这项研究中，我们使用了 4 种基于清洁剂的方法来脱细胞化 PAV，并旨在评估它们在去除抗原性细胞成分方面的效果。
使用十二烷基硫酸钠（SDS）、SDS 与脱氧胆酸钠（SDS/SD）、Triton X-100 和 Triton X-100 与 SD（Triton X-100/SD）分别对猪主动脉瓣进行脱细胞化处理。未处理的 PAV 用作对照。通过免疫组织化学和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析来确定抗原性蛋白成分的去除情况。通过组织学、生物化学和生物力学分析来确定细胞外基质的保存和机械性能。将 PAV 组织皮下植入 Sprague Dawley 大鼠体内，以评估宿主免疫反应。在指定的时间点取出植入的 PAV 进行组织学和免疫组织化学检查。
所有 4 种方案均能有效去除膜抗原性蛋白主要组织相容性复合体 I 分子和半乳糖-α-1,3 半乳糖。SDS/SD 方案是去除细胞质细胞骨架蛋白波形蛋白和α-SMA 的最有效方法，而 SDS 单独部分去除了波形蛋白蛋白。SDS/SD 方案是去除核 DNA 的最有效方法，残留 DNA 低于 50ng/mg，其次是 SDS 方案，残留 DNA 为 74.9ng/mg。SDS 方案是去除 10 至 55kDa 范围内蛋白质的最有效方法，其次是 SDS/SD 方案。术后第 3 天，SDS 和 SDS/SD PAV 植入物体内吸引的中性粒细胞较少。在第 14 天和第 28 天，SDS 和 SDS/SD 植入物中巨噬细胞和 T 淋巴细胞的浸润明显减少。与对照组相比，所有经过测试的脱细胞化方案都大大降低了胶原蛋白、弹性蛋白和糖胺聚糖的含量。生物力学分析显示，与 SDS、SDS/SD、Triton X-100 或 Triton X-100/SD 生成的对照 PAV 相比，SDS 生成的脱细胞 PAV 的极限拉伸强度和杨氏模量有显著差异。
这些结果表明，与 Triton X-100 相比，基于 SDS 的方案更有效地去除了抗原性细胞成分。这些结果具有临床意义，因为在异种应用中，完全去除抗原决定簇对于降低对 PAV 的不良免疫介导和炎症反应至关重要。
