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在体制备大鼠全肾去细胞支架的程序性分析
Programmed analysis of the method for the decellularization of intact rat kidney in vivo
目的 通过在体灌注Triton X-100、SDS溶液法制备大鼠全肾去细胞生物支架，并对支架制备过程中的关键步骤进行程序性检测、分析，为制备科学合理的实验用大鼠全肾去细胞生物支架提供基础。方法 SD大鼠40只，随机分为4组，每组10只。肝素化后直接切取肾脏作为对照组（control组）；肝素PBS灌注组（H组）；肝素PBS、Triton X-100灌注组（HT组）；肝素PBS、Triton X-100、十二烷基硫酸钠（SDS）溶液灌注组（HTS组）。HE、Masson、PAS染色及透射电镜观察各组肾组织病理及超微结构改变，免疫荧光结合4，6-二脒基-2-苯基吲哚（DAPI）观察各组胶原蛋白Ⅳ（collagen Ⅳ）、层黏连蛋白（LN）、纤维连接蛋白（FN）、硫酸乙酰肝素蛋白多糖2（HSPG2）、弹性蛋白（elastin）及细胞核的表达情况，总DNA测定各组DNA残留浓度。 结果 Triton X-100、SDS灌注6h左右制备大鼠肾脏去细胞生物支架。在灌注过程中，肾内细胞和细胞碎片逐渐被清洗，最终变成半透明状。HE、Masson、PAS染色及透射电镜显示连续分布、形态排列类似肾小球、肾小管轮廓结构的网状结构，基膜连续完整。免疫荧光结合DAPI染色结果表明，去细胞过程中细胞外基质中的重要蛋白collagen IV、LN、FN、HSPG2、elastin都较好的得到了保存，细胞核在灌注过程中逐渐减少，直至完全消失；最终残留组织的DNA为4.90μg/g。 结论 通过合适浓度和灌注比例的Triton X-100、SDS制备大鼠肾脏去细胞生物支架，并对其进行程序性分析，发现能有效清除大鼠肾内所有细胞成分，较完整的保留网络状肾及肾血管细胞外基质结构和成分，是一种简单易行且较为理想的制备实验用全肾生物支架的方法。
Objective To provide the underlying data for a new ideal method that can keep the intact rat kidney bioscaffold. Methods Decellularized rat kidney scaffolds was prepared by treating with Triton X-100, SDS sequentially in vivo and each steps of the method was observed separately.Forty SD rats were assigned to four groups randomly with 10 rats group. Normal rat kidneys were treated as control group after heparinization (CG), and then kidneys were perfused with 500ml heparinized PBS as H group, heparinized PBS and 1% Triton X-100 sequentially as HT group,heparinized PBS and 1% Triton X-100 and 1% SDS sequentially as HTS group. After each step of decellularization, the genomic DNA contect analysis, transmission electron microscopy, HE, Masson’s, PAS staining and immunohistochemistry techniques were used in order to observe changes in extracellular matrix constitution. Results Rat kidneys were decellularization in 6 hours. Its’cells and debris were cleaned gradually in the perfusion process and become translucent eventually. HE, Masson’s, PAS staining and transmission electron microscopy showed that the normal rat kidney was changed into reticular structure, it appeared to be continuous distribution and morphological arrangement was similar to the glomerular and tubular contours, and the integrity of it’s basement membrane was continuous. Immunofluorescence and DAPI staining showed that collagen IV, LN, FN, HSPG2, elastin were maintained during decellularization process. The DNA content in the scaffold was measured in order to demonstrate if there was the cells in the scaffold. The agarose gel electrophoresis showed that DNA bands were less in HTC group than those in control, H,HT groups. Conclusion The study shows that decellularized kidney maintains critical three-dimensional structure and contents by treating with Triton X-100, SDS sequentially at appropriate concentration and ratios. This method is ideal for the experiment of kidney tissue engineering and repair.
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