改良灌注法猪全胰腺脱细胞基质支架体外功能的实验研究

The research of porcine improved perfusion-acellular pancreatic bioscaffold as a natural 3D platform in vitro

目的采用改良灌注法制备猪全胰腺脱细胞基质支架(APB),并评价其生物学特征。方法选取10只新鲜的来自健康4~6月龄体重(20.0±2.5)kg猪的胰腺组织,采用本实验室自行设计的简易灌流装置制备猪APB,观察APB的形态结构,检测其细胞外基质(ECM)的成分以及DNA含量;随后采用移植实验评估APB生物相容性,最后将APB再细胞化观察其促进骨髓间充质干细胞(BMSCs)的增殖分化状况。结果脱细胞过程结束后APB颜色透明,原有组织轮廓保留完整;形态学检测表明APB保留原有的三维结构,ECM结构及成分保存较好,未见残留的细胞结构及成分;与正常猪胰腺组织相比,APB的ECM含量[胶原:(846.1±280.0)ng/mg干重比(754.7±249.6)ng/mg干重;葡萄糖胺聚糖(433.6±142.6)ng/mg干重比(397.2±98.7)ng/mg干重],差异无统计学意义(均P>0.05);但APB的DNA含量较正常猪胰腺组织降低[(83464.2±27813.4)ng/mg干重比(298.9±96.8)ng/mg干重,t=11.56,P<0.001],差异有统计学意义;APB在皮下埋植后随着时间延长,重塑评分升高,引起有益的重塑反应;再细胞化的APB可促进BMSCs的增殖及分化。结论本研究所制备的APB符合脱细胞标准,维持原有的三维结构,具有良好的生物相容性,有利于种植细胞的增殖分化。

Objective To investigate the biochemical properties of porcine improved perfusion-acellular pancreatic bioscaffold(APB).Methods The fresh pancreas from 10 health pigs[age:4-6 months,weight:(20.0±2.5)kg]were perfused through portal vein and pancreatic duct by our Easy-Load Digital Drive peristaltic pumps.The histological structure,extracellular matrix(ECM)composition and DNA quantification of APB were evaluated.The biocompatibility of APB was evaluated by transplantation experiment.Also,we observed the proliferation and differentiation of bone mesenchymal stem cells(BMSCs)reseeded on the APB.Results The pancreatic tissue became translucent and the macroscopic three-dimensional architectures of native pancreas were retained after decellularization.The histological evaluation indicated that the ECM ultrastructure was preserved,and there was no cellular components retained.Compared with the native pancreas,the major ECM components didn’t show significant difference[collagen:(846.1±280.0)ng/mg dry weight vs(754.7±249.6)ng/mg dry weight;sGAG:(433.6±142.6)ng/mg dry weight vs(397.2±98.7)ng/mg dry weight;both P>0.05].The DNA content of APB decreased significantly compared with native pancreas[(83464.2±27813.4)ng/mg dry weight vs(298.9±96.8)ng/mg dry weight,t=11.56,P<0.001].The histological analysis of the subcutaneous implantation site showed the enhanced histological remold score and benefit remodeling results with time after implantation.The proliferation and differentiation of BMSCs could be promoted on the recellularized APB.Conclusion Our APB in this study meets the stringent requirement to define a successful decellularization,which could allow APB preserving 3D architecture and represented a biocompatible scaffold capable of supporting and enhancing cell proliferation and differentiation.