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目的通过离体灌注加浸泡的方法制备大鼠胰腺去细胞生物支架(PDBC),为胰岛和胰腺的组织工程研究提供新型天然生物支架。方法健康成年大鼠20只,以物理冻融及灌注洗脱法(脱氧胆酸钠+DNAase),采用胆管、胰管联合逆向在体灌流法获取胰腺去细胞生物支架。并通过基因组DNA定量定性分析,组织化学染色、免疫荧光组织化学染色、荧光积分吸光度分析、透射电镜观察等进一步测定细胞残留以及观察去细胞支架,如胶原IV、纤维连接蛋白和层黏连蛋白等成分的保留。结果 DNA定性分析显示,实验组未见明显DNA条带,对照组DNA条带可达100bp,定量检测实验组DNA残留不足对照组的5%;组织化学染色和透射电镜观察均表明,去细胞支架无细胞残留,细胞外支架连续性完好,脉管支架保存完整;免疫荧光结果表明,胶原蛋白、弹性蛋白等细胞外支架成分保留较完整,未见明显细胞核成分残留。结论运用冻融加浸泡灌注制备的胰腺去细胞生物支架,细胞去除彻底,细胞外支架保留完好。
OBJECTIVE: To prepare rat pancreatic acellular biological scaffolds (PDBCs) by ex vivo perfusion and soaking, and to provide new natural biological scaffolds for the tissue engineering of islets and pancreas. Methods Twenty healthy adult rats were randomly divided into four groups: normal control group (n = 20) and normal control group (n = 20). The cell residue was further determined by quantitative and qualitative analysis of genomic DNA, histochemical staining, immunofluorescence histochemical staining, fluorescence integral absorbance analysis and transmission electron microscope observation, and the observation of decellularized scaffolds such as collagen IV, fibronectin and laminin Preservation of ingredients. Results DNA qualitative analysis showed that there was no obvious DNA band in the experimental group, DNA band in the control group was up to 100bp, and 5% of the DNA residue in the experimental group was quantitatively detected. The results of histochemical staining and transmission electron microscopy showed that the decellularized scaffold No cell residue, excellent continuity of extracellular scaffolds and intact vascular stents. Immunofluorescence results showed that extracellular scaffolds, such as collagen and elastin, remained relatively intact with no obvious nuclear components. Conclusion The preparation of pancreatic acellular scaffolds by freeze-thawing and soaking perfused the cells, the cells were completely removed and the extracellular scaffolds remained intact.