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      • KCI등재

        Severe choline deficiency induces alternative splicing aberrance in optimized duck primary hepatocyte cultures

        Zhao Lulu,Cai Hongying,Wu Yongbao,Tian Changfu,Wen Zhiguo,Yang Peilong 아세아·태평양축산학회 2022 Animal Bioscience Vol.35 No.11

        Objective: Choline deficiency, one main trigger for nonalcoholic fatty liver disease (NAFLD), is closely related to lipid metabolism disorder. Previous study in a choline-deficient model has largely focused on gene expression rather than gene structure, especially sparse are studies regarding to alternative splicing (AS). In modern life science research, primary hepatocytes culture technology facilitates such studies, which can accurately imitate liver activity in vitro and show unique superiority. Whereas limitations to traditional hepatocytes culture technology exist in terms of efficiency and operability. This study pursued an optimization culture method for duck primary hepatocytes to explore AS in choline-deficient model. Methods: We performed an optimization culture method for duck primary hepatocytes with multi-step digestion procedure from Pekin duck embryos. Subsequently a NAFLD model was constructed with choline-free medium. RNA-seq and further analysis by rMATS were performed to identify AS events alterations in choline-deficency duck primary hepatocytes. Results: The results showed E13 (embryonic day 13) to E15 is suitable to obtain hepatocytes, and the viability reached over 95% by trypan blue exclusion assay. Primary hepatocyte retained their biological function as well identified by Periodic Acid-Schiff staining method and Glucose-6-phosphate dehydrogenase activity assay, respectively. Meanwhile, genes of alb and afp and specific protein of albumin were detected to verify cultured hepatocytes. Immunofluorescence was used to evaluate purity of hepatocytes, presenting up to 90%. On this base, choline-deficient model was constructed and displayed significantly increase of intracellular triglyceride and cholesterol as reported previously. Intriguingly, our data suggested that AS events in choline-deficient model were implicated in pivotal biological processes as an aberrant transcriptional regulator, of which 16 genes were involved in lipid metabolism and highly enriched in glycerophospholipid metabolism. Conclusion: An effective and rapid protocol for obtaining duck primary hepatocytes was established, by which our findings manifested choline deficiency could induce the accumulation of lipid and result in aberrant AS events in hepatocytes, providing a novel insight into various AS in the metabolism role of choline. Objective: Choline deficiency, one main trigger for nonalcoholic fatty liver disease (NAFLD), is closely related to lipid metabolism disorder. Previous study in a choline-deficient model has largely focused on gene expression rather than gene structure, especially sparse are studies regarding to alternative splicing (AS). In modern life science research, primary hepatocytes culture technology facilitates such studies, which can accurately imitate liver activity <i>in vitro</i> and show unique superiority. Whereas limitations to traditional hepatocytes culture technology exist in terms of efficiency and operability. This study pursued an optimization culture method for duck primary hepatocytes to explore AS in choline-deficient model.Methods: We performed an optimization culture method for duck primary hepatocytes with multi-step digestion procedure from Pekin duck embryos. Subsequently a NAFLD model was constructed with choline-free medium. RNA-seq and further analysis by rMATS were performed to identify AS events alterations in choline-deficency duck primary hepatocytes.Results: The results showed E13 (embryonic day 13) to E15 is suitable to obtain hepatocytes, and the viability reached over 95% by trypan blue exclusion assay. Primary hepatocyte retained their biological function as well identified by Periodic Acid-Schiff staining method and Glucose-6-phosphate dehydrogenase activity assay, respectively. Meanwhile, genes of <i>alb</i> and <i>afp</i> and specific protein of albumin were detected to verify cultured hepatocytes. Immunofluorescence was used to evaluate purity of hepatocytes, presenting up to 90%. On this base, choline-deficient model was constructed and displayed significantly increase of intracellular triglyceride and cholesterol as reported previously. Intriguingly, our data suggested that AS events in choline-deficient model were implicated in pivotal biological processes as an aberrant transcriptional regulator, of which 16 genes were involved in lipid metabolism and highly enriched in glycerophospholipid metabolism.Conclusion: An effective and rapid protocol for obtaining duck primary hepatocytes was established, by which our findings manifested choline deficiency could induce the accumulation of lipid and result in aberrant AS events in hepatocytes, providing a novel insight into various AS in the metabolism role of choline.

      • KCI등재

        Effect of curing conditions on mode-II debonding between FRP and concrete: A prediction model

        Pengcheng Jiao,Sepehr Soleimani,Quan Xu,Lulu Cai,Yuanhong Wang 사단법인 한국계산역학회 2017 Computers and Concrete, An International Journal Vol.20 No.6

        The rehabilitation and strengthening of concrete structures using Fiber-Reinforced Polymer (FRP) materials have been widely investigated. As a priority issue, however, the effect of curing conditions on the bonding behavior between FRP and concrete structures is still elusive. This study aims at developing a prediction model to accurately capture the mode-II interfacial debonding between FRP strips and concrete under different curing conditions. Single shear debonding experiments were conducted on FRP-concrete samples with respect to different curing time t and temperatures T. The J-integral formulation and constrained least square minimization are carried out to calibrate the parameters, i.e., the maximum slip s ̅ and stretch factor n. The prediction model is developed based on the cohesive model and Arrhenius relationship. The experimental data are then analyzed using the proposed model to predict the debonding between FRP and concrete, i.e., the interfacial shear stress-slip relationship. A Finite Element (FE) model is developed to validate the theoretical predictions. Satisfactory agreements are obtained. The prediction model can be used to accurately capture the bonding performance of FRP-concrete structures.

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