| BackgroundLiver diseases generally result in hepatic failure,for which orthotopic liver transplantation is the only definitive treatment and the most effective therapy.However,the major issue remains that there is limited numbers of donor livers for transplantation.Therefore,a significant advancement has been the utilization of decellularized liver bioscaffold(DLB)as a three-dimensional scaffold in tissue engineering.Upon the DLB,recellularized liver is generated by reseeding a variety of seed cells.As one approach in hepatic tissue engineering,recellularized liver is an effective alternative to liver transplantation for end-stage hepatic failure.However,there is still disparity for recellularized liver when compared with normal liver.Though sympathetic nerve is considered as an important constituent in liver and innervates the process of physiology and pathology in liver.It is still unkown that whether sympathetic neurotransmitters play a role in regulation of recellularized liver.ObjectiveToexplore the effect of activation of adrenergic receptors on hepatocytes in DLB and hepatic function of recellularized liver.And to discuss what mechanismmay be involved in this process.Methods1.DLB was achieved by whole-organ decellularization and analyzed by nucleotide detection and histological assessment,and recellularized liver was generated and analyzed by immunofluorescence.2.The expressions of subtypes of adrenergic receptors(ARs)on hepatocyte were screened by RT-PCR and immunofluorescence.3.The effects of specific AR agonists on cell proliferation both in the plain and in recellularized liver were observed by Cell Titer-Blue method and Click-i T Plus EdU staining.4.The effects of specific AR agonists on albumin secretion and urea synthesis in recellularized liver were analyzedby Elisa to detect the hepactic function of recellularized liver.5.Mechanism of the promotive process of effective AR agonist was clarified by RT-PCR and Western Blot analysis.Results1.DLB and recellularized liver were successfully generated and analyzed.Vascular tree was clearly visible under low fluorescence gross microscopy.H&E staining showed no nuclear or cytoplasmic staining in the DLB compared with normal liver,while identifiable cells were clearly seen in the recellularized liver.Massion staining revealed that both the structure and components of the DLB and recellularized liver were retained similarly to normal mouse liver.Spectrophotometric analysis indicated the removal of 99% of the DNA from the normal liver.The quantity of cells increased over time in the L02-seeded bioscaffold.2.Specific subtypes of ARs were expressed on the hepatocyte membrane.RT-PCR showed that expression level of AR-?1D and AR-?2 was significantly high,and AR-?1A and AR-?2B were almost undetectable.Immunofluorescence showed expression level of AR-?2 was significantly high in the membrane.3.Salbutamol,agonist of AR-?2,promoted cell proliferation,albumin secretion and urea synthesis in recellularized liver.4.Signal molecules were screened in isoproterenol/salbutamol treated recellularized liver,and expression of IL-6 significantly increased.5.Isoprenaline/salbutamol particularly promoted the expressions of Stat3 and phosphorylated Stat3,but not those of Smad3,p38,JNK-1,and Akt-1,and contributed to the activation of IL-6/Stat 3 signaling in promoting hepatocyte proliferation and hepatic function of recellularized liver.ConclusionThis study suggested that activation of AR-?2 accelerated hepatocyte proliferation in recellularized liver and improved recellularized liver function by mediating IL-6/Stat 3 signal pathway,indicating that sympathetic neurotransmittersmay be essential supplements to complexity of recellularized liver in tissue engineering. |
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