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目的:通过不同脱细胞方法制备脱细胞血管基质材料,比较筛选出较为理想的血管脱细胞方法,为临床血管修复提供良好的组织工程生物材料。方法:1采用不同浓度的胰蛋白酶、Triton X-100、SDS组合,对牛血管进行脱细胞处理,制备脱细胞牛血管基质。2苏木素-伊红染色,观察细胞核残留情况,以比较脱细胞效果。3利用微量样品基因组DNA提取试剂盒提取脱细胞血管基质的DNA,超微量蛋白核酸分析仪检测DNA含量。结果:大体观察:各组血管经过不同脱细胞方法处理后,所得脱细胞血管基质呈乳白色。苏木素-伊红染色观察:光镜下观察,可见各组均有不同程度的细胞核残留,其中1%胰蛋白酶+3%Triton X-100+1%SDS组和0.1%胰蛋白酶Triton X-100+0.5%SDS所制备的脱细胞血管基质细胞核残余最少,每高倍视野中细胞核残留不大于3。DNA提取检测:所提取DNA的含量显示,0.1%胰蛋白酶+3%Triton X-100+1%SDS和0.1%胰蛋白酶+3%Triton X-100+0.5%SDS的脱细胞效果较好,DNA残留量分别为0.22μg/mg和0.231μg/mg。结论:应用0.1%胰蛋白酶+3%Triton X-100+1%SDS和0.1%胰蛋白酶+3%Triton X-100+0.5%SDS的方法来制备脱细胞基质的效果较好。
OBJECTIVE: To prepare acellular vascular matrix material by different acellular methods and compare with the ideal method of acellular vascular decellularization to provide a good tissue engineering biomaterial for clinical vascular repair. Methods: 1 The bovine blood vessels were decellularized with different concentrations of trypsin, Triton X-100 and SDS to prepare acellular bovine vascular matrix. 2 hematoxylin-eosin staining, observe the nuclear residue, in order to compare the decellularization effect. 3 using a sample of genomic DNA extraction kit extracted acellular vascular matrix DNA, DNA content of DNA microarray detection. Results: In general, blood vessels from each group were milky white after treatment with different acellular methods. Hematoxylin-eosin staining: Observed by light microscopy, we can see that each group has different degrees of nuclear residues, including 1% trypsin + 3% Triton X-100 + 1% SDS group and 0.1% trypsin Triton X-100 + 0.5% SDS prepared acellular vascular stromal nuclear remnant minimum, each high power field in the nucleus of no more than 3. DNA Extraction Detection: The extracted DNA content showed better decellularization effect of 0.1% trypsin + 3% Triton X-100 + 1% SDS and 0.1% trypsin + 3% Triton X-100 + 0.5% Residues were 0.22 μg / mg and 0.231 μg / mg, respectively. CONCLUSION: Acellular matrix is best prepared using 0.1% trypsin + 3% Triton X-100 + 1% SDS and 0.1% trypsin + 3% Triton X-100 + 0.5% SDS.