离体大鼠胰腺去细胞生物支架的制备与鉴定

Preparation and identification of the pancreatic decellularized bio-derived scaffold in isolated rats

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 a pancreatic decellularized bio-derived scaffold（PDBC） in vitro, providing a novel natural bio-derived scaffold for islet and pancreatic tissue engineering. Methods The pancreas from adult SD rats was obained. To prepare the PDBC, we used physical freeze thawing, socking and perfusing that based on retrograde pancreatic and bile ducts＇ perfusion. After dccellularization, normal pancreatic tissue and PDBC were examined by genomic DNA quantification, and through HE staining, transmission electron microscope and immunohistochemistry. The conservation of collagen type IV, fibronectin and laminin were identified, Results The DNA gelelectrophoresis showed that no bands in the experimental group while a 100bp band appeared in the control group. Compared with the DNA in the control group, the DNA in PDBC was less than 5%. Histochemical staining and transmission electron microscopy indicated that PDBC preserved the complete extracellular matrix and no cells attached to PDBC surface. Immunofluorescence results demonstrated that collagen, elastin and other extracellular components were well preserved, without obvious nuclear components. Conclusion The PDBC prepared by freeze thawing, socking and perfusing was decellularized compietely and preserved the extracellular matrix fully.