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Programmed analysis of the method for the decellularization of intact rat kidney in vivo
ZHAO Li-na YU Ya-ling LIN Ke-zhi SHAO Ying-kuan WANG Zhi-bin CHEN Jia-jun MEI Jin* TANG Mao-lin*
Acta Anatomica Sinica ›› 2014 ›› Issue (2) : 267-272.
Programmed analysis of the method for the decellularization of intact rat kidney in vivo
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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