The Research Of Xenologous Decellularized Arterial Extracellular Matrix To Repair Biliary Epithelium And Biliary Duct Defect In Diannan Swine

Part One Preparation of decellularized arterial extracellular matrix of human and its histological featuresObjective:To choose hypothetical tissue-engineering scaffold for biliary tract defect repair by preparing decellularized arterial matrix and biliary tract matrix of human and observing histological features of these tissue scaffolds.Methods:Spleen artery, partial aorta and extrahepatic biliary tract were obtained from donor after cardiac death in Affiliated Calmette Hosptial of Kunming Medical University.1% Sodium dodecyl sulfonate(SDS),1% TritonX-100 and 0.25% Trypsin solution were used to prepare decellularized arterial and biliary tract matrix, hematoxylin and eosin(HE) staining and scanning electron microscope(SEM) were applied to observe histological features of decellularzied and normal tissues, the total DNA in normal and acellular tissue was quantified by spectrophotometer.Results:In HE staining and SEM samples, decellularzied arterial and biliary tract matrix maintained complete extracellular structure while in the absence of apparent cell structures, different layers of decellularzied spleen artery could be observed with complete collagen fibers. The intima thickness of acellular artery and biliary tract was 114.83±21.67μm and 19.87±4.52μm respectively, the difference was statistically significant (P=0.000). DNA quantity in acellular arterial and biliary tract matrix was less significantly than normal tissue (P=0.000, P=0.000)Conclusion:1%SDS,1% TritonX-100 and 0.25% Trypsin could decellularize artery and biliary tract effectively with complete extracellular collagen fibers, Thicker intima could be observed in acellular artery with better porosity than biliary tract, and compare to normal tissue, DNA quantity of acellular artery matrix was less than normal tissue. Because it is easier to harvest more artery in one single donor than extrahepatic biliary tract, decellularzied arterial matrix might be ideal tissue-engineering scaffold for biliary duct defect repair.Part Two Isolation, culture, identification of rat hepatic oval cell and induced it differentiate into cholangiocyte in vitroObjective:To obtain cholangiocytes as seed cells of tissue-engineering research, which derived from isolated and cultured rat hepatic oval cells(HOC) in vitro. Methods:2-acetaminofluorene (2-AAF) feeding and partial hepatectomy(PH) was used to establish activated HOC animal model in SD rat. Then liver tissue was harvested from animal model, Protease E was combined with collagenase Ⅳ to digestive liver tissue to obtain primary HOC. Epithelium growth factor (EGF), stem cell growth factor (SCF) and leukemia inhibitory factor(LIF) consisted of induced protocol to make HOC differentiation. Morphological features of HOC were observed in the process of culture, while HOC specific biomarker OV-6 and cholangiocyte specific marker CK-19 positive cells in different passages were measured by Flow cytometry and Immuno fluorescence microscopy (IFM). QRT-PCR was utilized to relative-quantified the CK-19, y-GT, AFP, ALB mRNA in different passages.Results:The primary cultured cell, which derived from SD rat liver tissue, was in accord with morphological characteristics of HOC, the OV-6 and CK-19 positive rate of primary cultured cell was 94.30±1.40% and 3.16±1.87% respectively, compared to cells in Passage 6, which included statistically significant difference in OV-6 and CK-19 positive rate cells 4.97±0.21% and 96.17±1.21%(P=0.000). IFM observation revealed that along with cell passage increase, the number of OV-6 positive cell decreased, but number of CK-19 positive cells added up. Meanwhile, the morphological changes of CK-19 positive cells conformed to process of HOCs differentiate into biliary epithelial cells. CK-19、γ-GT、AFP、ALB mRNA in the second, eighth, tenth and twelfth passage were relative-quantified, compare to the second passage, the Relative-quantity(RQ) of CK-19、γ-GT、AFP、ALB mRNA in twelfth passage was 5.3471±0.1057,3.4820±0.2393,0.1980±0.0131,0.6724±0.1700 respectively, all the differences were significant.Conclusion:HOCs were successfully induced into cholangiocytes by the protocol included EGF, SCF and LIF, in differentiation process, change of morphological features, biomarker and gene expression in accord with the characteristics of HOCs differentiate into cholangiocytes.Part Three The compatibility of acellular arterial matrix from human and induced cholangiocytes in vitroObjective:To observe growth of cholangiocytes and its compatibility with acellular arterial matrix by seeding induced cholangiocytes on decellularzied arterial matrix.Methods:Decellularzied arterial matrix was sliced into 1×1cm pieces, then induced cholangiocytes was digested and suspended in concentration of 5×106/ml.1×106 .cholangiocytes were seeded on the acellular arterial matrix, which had been immersed in DMEM/F12 medium for 24 hours. The complex of cholangiocytes and acellular arterial matrix was immersed into culture medium after put into incubator for 4 hours. 4,6 and 8 weeks later, acelllular arterial matrix was taken out, immunofluorecence microscope and scanning electron microscope were used to observe were the cholangiocytes grown well or not.Results:4 weeks after cholangiocytes were seeded on the decellularized arterial matrix, CK-19 positive cells could be seen by IFM.8 weeks later, 1×1cm acellular arterial matrix was covered completely by CK-19 positive cells, epithelium-like structure had been formed under scanning electron microscope observation. Conclusion: Decellularized arterial matrix from human was compatible with cholangiocytes, it is could be scaffold for cholangiocytes growth in vitro, it would take 8 weeks that cholangiocytes covered 1×1cm acellular arterial extracellular matrix.Part Four The experimental study about xenologous decellularized arterial matrix repair extrahepaitc biliary duct defect in Diannan swineObjective:To explore the effect and mechanism of xenologous arterial extracellular matrix on repairing extrahepatic biliary duct defect by using acellular arterial matrix to rebuild defect of common bile duct in Diannan swine.Methods:10 Diannan swine in closed colony was divided into 3 groups randomly: Group A(n=4), which accepted biliary duct repair operation by acellular arterial matrix; Group B(n=4), whose common bile ducts were transected before duct to duct anastomosis; Group C (n=2) was blank controlled group. In different postoperative times, venous blood samples were taken to examine total bilirubin (TBIL), alkaline phosphatase (ALP), and y-glutamyltranspetidase (y-GT), and differences between groups were compared.12 weeks after operation, all the experimental animals were executed to procure extrahepatic biliary tract samples. Hematoxylin and eosin (HE) staining was used to observe histological features of biliary duct. In the same time, immunohistochemistry(IHC) was applied to explore CK-19、TGF-β1、 CTGF、VEGF expression in bile ducts, and Image Pro Plus (IPP) software, which could transfer IHC pictures into gray ones, was used to compare differences in Integrated optical density (IOD) between groups. At last, acellular arterial matrix, which had been implanted into swine for 12 weeks, was examined by scanning electron microscope to survey condition of cholangiocyte on mucosa.Results:61 days after operation, one animal in Group A died, all the other animals survived in 12 weeks experiment. Compared to Group B and C, TBIL, ALP and y-GT of swine in Group A were significantly higher while IOD of CK-19、TGF-β1、 CTGF、 VEGF expression were different. Plenty of cells from recipient animal could be found in acellular arterial matrix, and a few newly formed blood vessels also could be seen. Cholangiocyte-liked cell were observed under scanning electron microscope, but epithelium was not integrated.Conclusion:12 weeks after extahepatic biliary tract repair of swine by decellularized arterial matrix from human, bile duct and its blood supply could be rebuilt partially, xenologous acellular arterial matrix was compatible with recipient Diannan swine, furthermore, cholangiocytes partially regenerated on the mucosa of arterial matrix. However, obvious differences could be seen between repaired bile duct by acellular arterial matrix and normal ones. Based on this experiment, decellularized arterial matrix could rebuild biliary duct structure partially, if functional reconstruction of biliary duct needs to be fulfilled in the future, more researches in decellularized arterial matrix should proceed.