| BackgroundCurrently, liver transplantation is the most definite curative for various liver diseases that lead to end-stage liver failure in the medical field. But, at present, the number of donor can not meet the demand of liver transplantation. So, how to solve the problem becomes a hot research topic in the field of organ transplantation.At present, decellularized liver bioscaffold(DLB) is a new point in tissue engineering which through decellular methods to realize the three-dimensional reconstruction of liver structure, retaining the original most of the extracellular matrix components, including collagen and elastic fiber. Trying to construct an ideal DLB, solving the shortages of the traditional scaffolds which low immunogenicity, bionic resistance and mechanical strength of the weak, and eventually the cell material of liver cells and endothelial cells is related to biological scaffold materials which for cells of liver to realize complex, building for graft tissue organ in vitro, and realizing the morbid organs the partial or complete replacement.The above process is a milepost field of tissue engineering and organ transplantation. ObjectiveIn this experiment, combined with the previous scheme for preparing decellularized organs, using systemic methods to perfusion device combined by chemical and enzymatic that is designed to prepare the rat decellularized bioscaffold, carrying on the composition analysis after the preparation of bioscaffold, and providing experimental data for the next step which replant seed cells for construction of artificial liver. Methods1.Adult SD rats, male or female, anaesthetize with 10% chloral hydrate intraperitoneal injection. Take abdominal longitudinal incision, up to the xiphoid process, next to the upper edge of pubic symphysis separation, separating the liver and surrounding tissue carefully. In the inferior vena cava and right renal vein confluence, for systemic heparinization with the 200U/ml heparin sodium solution. Placing a 20 G indwelling needle in the portal vein under the micromanipulation, cutting the liver and hepatic inferior vena cava, the stroke-physiological saline solution by indwelling needle injection in the liver, liver color gradually becomes yellow soil, then stop infusion. Separating the liver tissue around the remaining intact, removing the liver carefully.2.Livers were divided into control group and experiment group, the control group of 4% formalin fixed and retained, the experiment group was implanted with acellular device, firstly by compounding ratio of 0.02% trypsin and 0.05%EDTA mixed solution perfusion 2h, secondly by compounding ratio of 4%Triton-X 100 and 0.05%EDTA mixed solution perfusion 20-24 h, and then use the 1%SDS solution perfusion of 2h, then the compounding ratio of 0.1% peracetic acid and 4% ethanol mixed solution perfusion 2h, and finally with cefazolin sodium solution perfusion of liver 6h, the decellularized liver scaffold is successful made.3.The control group and the experiment group were fixed, routine HE staining, transmission electron microscope scanning, at the same time for the analysis of collagen type I, CD35, LCA and Vimentin protein immunohistochemistry. Results1.Observation of rat liver color gradually changes from yellow to translucent during perfusion, vascular system of the rat liver gradually appeared.2.After HE staining, the extracellular matrix of control group showed a lot of blue and dense nuclei and pink matrix, has obvious fracture among the collagen fibers. DLB has no obvious nuclear component residue, in the extracellular matrix can be observed much structure and dense pink matrix.3.Cell structure in the control group is obvious via transmission electron microscope scanning, including clear nuclei, dictyosome, endoplasmic reticulum, the residual DLB by transmission electron microscope scan showed no obvious cell structures, a lot of fiber woven net structure, arranged in an orderly manner, at the same time there are the tubular like structures.4.Pairs of type I collagen, the control group and the DLB CD35, LCA and Vimentin protein immunohistochemical(IHC)OD results were compared, CD35, LCA and Vimentin protein in control group and DLB had significant difference, type I collagen in contrast group and in DLB has no statistical significance. ConclusionsThe use of chemical and enzymatic for perfusion decellularized scaffold can be constructed in rats, the scheme can effectively remove the cellular components in the liver, retenting the extracellular matrix, the DLB can be used for the study of liver tissue engineering. |
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