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大鼠胰腺去细胞天然支架生物相容性的鉴定
邵营宽 严夏霖 饶志恒 黄高建 李嘉威 黄俊杰 梅劲* 林刻智*
解剖学报 ›› 2014, Vol. 45 ›› Issue (4) : 561-568.
大鼠胰腺去细胞天然支架生物相容性的鉴定
Biocompatibility of rat’s nature decellularized pancreatic biological scaffolds
目的 通过离体灌注化学去垢剂的方法制备大鼠胰腺去细胞天然生物支架，并对支架的完整性、生物相容性进行检验。方法 健康成年SD大鼠30只，分别经胆管与血管两条途径灌注十二烷基硫酸钠和曲亚通X-100等药品洗脱，获取胰腺去细胞生物支架。通过HE染色、扫描电子显微镜和透射电子显微镜等观察细胞残留于去细胞支架的生物学特性，分光光度计法鉴定残留去垢剂量，酶联免疫吸附剂测定法测定残留支架蛋白含量及生长因子，并用腹壁与皮下包埋实验检验其炎症反应，MTT法测定支架细胞毒性，内皮细胞共培养测定支架生物相容性。 结果 HE染色、扫描电子显微镜和透射电子显微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；药物残留小于规定标准；细胞外支架生长因子及支架蛋白存留量等指标上血管组均优于胆管组；腹壁与皮下包埋实验显示，去细胞支架炎性反应较对照组明显减轻；MTT法显示无细胞毒性；内皮细胞共培养有黏附趋势。结论 使用两种灌注法制备胰腺去细胞生物支架，均可将细胞去除彻底，支架生物相容性好。但就血管组细胞外支架保留完整性，生长因子保留更多，则血管灌注途径优于胆管灌注途径。
Objective To harvest pancreatic tissues from rats, prepare decellularized bio-derived pancreatic scaffolds (DBPS), and to examine the integrity and biocompatibility of the scaffolds. Methods Normal pancreases were harvested from healthy adult SD rats. DBPS was prepared by perfusing SDS and Triton X-100 through bile duct and the portal vein, respectively. After decellularization, normal pancreatic tissue and DBPS were compared via HE staining, and transmission electron microscopy (TEM). Abdominal wall and subcutaneous implantations were used to compare biocompatibility, and the remain quantity of residual protein and growth factors were determined via enzyme linked immunosorbent assay（ELISA）. MTT assay was used to test the scaffolds’ cytotoxicity. The scaffolds were co-cultured with endotheliocyte. Results HE staining and TEM study indicated no residual cells in the DBPS as well as preservation of the complete extracellular matrix. The remain quantity of residual protein and growth factors in ECM was high. The abdominal wall and subcutaneous implantation revealed that DBPS triggered a lower immune response as compared to the control group. MTT assay showed little cytotoxicity. Endotheliocyte assembled and growed with the scaffolds together.
Conclusion DBPS are completely decellularized, and exhibit a higher level of biocompatibility in vivo. Using the way of vessels can make the integrity of extracellular matrix to be fully preserves and contain more growth factors. So using vessels way is better than bile duct.
去细胞 / 胰腺 / 细胞外基质 / 生物相容性 / 组织工程 / 大鼠
Decellularization / Pancreas / Extracellular matrix / Biocompatibility / Tissue engineering / Rat
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国家级大学生创新创业训练计划项目;浙江省大学生科技创新活动计划“新苗人才计划”;浙江省自然科学基金
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