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目的:证实去除细胞外基质蛋白多糖对提高脱细胞猪肺动脉带瓣管道抗钙化性能的作用,为研制组织工程化肺动脉带瓣管道做准备。方法:实验分为3组,即A组:为新鲜猪肺动脉带瓣管道组织,B组:用胰蛋白酶+Triton X-100处理的脱细胞猪肺动脉带瓣管道组织和C组:在B组处理的基础上再经透明质酸酶消化,去除细胞外蛋白多糖基质成分的猪肺动脉带瓣管道组织,每组4份(n=4)。方法:实验室:将3组样本分别进行HE染色后,用光镜和扫描电镜观察肺动脉管壁及瓣膜组织的变化。采用盐酸胍抽提结合阿利新蓝染色法测定蛋白多糖的含量。同时将3组样本包埋于大鼠皮下,于6周后取出标本进行Van Kosaa银染色法(钙盐染色)和原子吸收分光光度计法分别定性、定量分析组织的钙化程度。结果:光镜和电镜检查结果显示,猪肺动脉管壁和瓣膜组织的细胞可以较完整的去除,纤维网架结构可以完整保持。蛋白多糖含量的测定显示,与A、B组相比,C组细胞外基质蛋白多糖的含量显著下降(P<0.05)。大鼠皮下包埋实验显示,与A、B两组相比,C组的钙化反应更少,管壁组织钙的含量显著下降(P<0.05)。结论:采用胰蛋白酶+Triton X-100脱细胞方法可以达到去除细胞的目的。通过大鼠皮下包埋实验证明,采用透明质酸酶消化减少细胞外基质蛋白多糖的含量可以进一步减少脱细胞组织的钙化反应,为组织工程肺动脉带瓣管道的构建提供较为理想的脱细胞基质材料。
OBJECTIVE: To confirm the effect of extracellular matrix proteoglycan on anti-calcification of aortic valve in acellular porcine pulmonary artery and to prepare for the tissue engineering of pulmonary valve. Methods: The experiment was divided into three groups, namely, group A: fresh pulmonary artery of pulmonary valve tissue; group B: aorta pulmonary artery valve tissue treated with trypsin + Triton X-100 and group C: group B (N = 4), followed by hyaluronidase digestion to remove the extracorporeal proteoglycan matrix components of the pulmonary artery of the pulmonary valve tissue. Methods: Laboratory: The three groups of samples were HE staining, the light microscope and scanning electron microscopy of pulmonary artery wall and valve tissue changes. The content of proteoglycan was determined by guanidine hydrochloride extraction combined with Aliskiren staining. At the same time, three groups of samples were subcutaneously embedded in the rat. After six weeks, the specimens were removed and qualitatively and quantitatively analyzed for the degree of calcification by Van Kosaa silver staining and atomic absorption spectrophotometer respectively. Results: The results of light and electron microscopy showed that the cells of the pulmonary artery wall and the valve tissue of the porcine pulmonary artery could be completely removed, and the structure of the fibrous scaffolds could be completely preserved. The content of proteoglycan showed that the content of extracellular matrix proteoglycan in group C was significantly lower than that in group A and B (P <0.05). Subcutaneous embedding experiments in rats showed that compared with A and B groups, the calcification reaction in C group was less and the content of calcium in tube wall tissue was significantly decreased (P <0.05). Conclusion: The removal of cells can be achieved by trypsin + Triton X-100 acellular method. Subcutaneous burial experiments in rats demonstrated that the use of hyaluronidase to reduce the extracellular matrix proteoglycan content can further reduce the calcification reaction of acellular tissue, tissue engineering for the construction of pulmonary artery valve with flap provide a more ideal acellular matrix material .