猪胆总管脱细胞支架的制备及组织学评价

Preparation and histological evaluation of the decellularized scaffold for porcine common bile duct

目的用不同的脱细胞方法对猪胆总管进行处理,比较脱细胞前后的组织学变化,筛选适宜的脱细胞方法,为组织工程胆管支架材料的应用提供理论依据。方法 30例猪胆总管随机分为5组:对照组(A组):0.05%胰蛋白酶+核酸酶(B组):0.1%十二烷基硫酸钠(SDS)+核酸酶(C组):1.0%Triton X-100+核酸酶(D组):1.0%Triton X-100+0.1%SDS+核酸酶(E组)。通过HE染色光学显微镜下观察脱细胞基质的组织结构和细胞残留情况;用紫外分光光度法检测脱细胞基质的DNA含量,计算脱细胞率。结果脱细胞B组有少量细胞残留,纤维有损伤;脱细胞C组、D组、E组的细胞均被去除,纤维无明显损伤。A组的DNA含量为(71.24±2.56)μg/100 mg。B、C、D、E4个脱细胞组的DNA含量与A组的差异有统计学意义(F=15.29,P<0.01),均有明显的脱细胞效果(P<0.01)。B组的脱细胞率为77.03%,比C组、D组、E组的脱细胞效果稍差(P<0.05),E组的脱细胞率高达99.03%。结论应用1.0%Triton X-100+0.1%SDS+核酸酶的脱细胞效果好,能更好地降低胆总管的免疫原性,是一种比较理想的猪胆总管脱细胞方法。

Objective To evaluate the histological change of porcine common bile ducts before and after treatments with different acellular matrix, and to provide theoretical basis for the application of bile duct scaffold materials for tissue engineering. Methods Thirty porcine common bile ducts were randomly divided into 5 groups： control （A） group： 0.05% trypsin ＋ nuclease （B）, 0. 1% sodium dodecyl sulphate （SDS） ＋ nuclease（C） , 1.0% triton X- 100 ＋ nuclease（D）, 1.0% triton X - 100 ＋ 0. 1% SDS ＋ nuclease（E） groups. After HE staining, the tissue structure of acellular matrix and the residual cells were observed under a light microscope. The DNA content of acellular matrix was detected using UV spectrophotometry, and the acellular rate was calculated. Results Few residual cells and fiber damage were observed in group B. All the cells were removed and no obvious damages were done in groups C, D and E. The DNA content of group A was （71.24 ± 2.56） μg / 100 mg. The DNA contents of four acellular matrix treatment groups B, C, D and E were statistically different from that of group A （ F = 15.29, P 〈0.01 ）. There was obvious acellular matrix treatment effect in the four groups （P 〈 0.01 ）. The acellular rate of group B was 77.03% , the acellular matrix treatment effect of group B was slightly worse that of groups C, D and E （P 〈 0. 05）. The acellular rate of group E was up to 99.03%. Conclnsion The acellular matrix treatment effect of 1.0% Triton X-100 ＋ 0.1% SDS ＋ nuclease is satisfactory. The immunogenicity of the common bile duct can be reduced, therefore, it is an ideal acellular method for porcine common bile ducts.