Establishment and functional in vitro characteristics of three-dimensional collagen HepaRG microsphere / 中国组织工程研究

BACKGROUND: Some recent studies have shown that the three-dimensional (3D) model of HepaRG cells can better mimic the in vivo microenvironment and show better liver differentiation and function compared with two-dimensional culture. OBJECTIVE: HepaRG was selected to prepare 3D collagen microspheres, and the adaptive culture and functional expression of cells in the collagen microspheres were evaluated. METHODS: Collagen hydrogel was used as the scaffold for 3D HepaRG and HepG2 microspheres. Stable cell spheres were formed. HepaRG microspheres, HepG2 microspheres, HepaRG two-dimensional culture and HepG2 two-dimensional culture were used as controls. At 1, 6, and 12 days of culture, cell survival was detected by the Live/Dead assay staining. After 1, 6, 12, and 16 days of culture, the urea synthesis and CYP3A4 secretion of the supernatant were detected in each group. After 12 days of culture, relative expression of CYP3A4, CYP1A2, UGT1A1, and CPS1 mRNA was detected by qPCR. The expression levels of hepatocyte marker albumin and CYP3A4 protein were determined using western blot assay. RESULTS AND CONCLUSION: (1) In 12 days of culture, Live/Dead assay staining showed that the cell viability in the 3D collagen microsphere was well-maintained and the amount of central necrotic cells was small, with high cell viability. In the 3D collagen microsphere, especially HepaRG cells, multiple cellular clusters formed and adjacent clusters were connected closely, which created a cross-linked structure. (2) After 1, 6, and 12 days of culture, the urea content of HepaRG 3D collagen microspheres was higher than that of HepaRG two-dimensional culture (P < 0.05). After 1, 6, 12, and 16 days of culture, the urea content of HepG2 3D collagen microspheres was higher than that of HepG2 two-dimensional culture (P < 0.05). After 1, 6, and 12 days of culture, the secretion of CYP3A4 in HepaRG 3D collagen microspheres was higher than that in HepaRG two-dimensional culture (P < 0.05). After 6 and 12 days of culture, the secretion of CYP3A4 in HepG2 3D collagen microspheres was higher than that in HepG2 two-dimensional culture (P < 0.05). (3) The relative expression of CYP3A4, CYP1A2, UGT1A1, and CPS1 mRNA in HepaRG 3D collagen microspheres was higher than that in HepaRG two-dimensional cells (P < 0.05), and the relative expression of CYP1A2 in HepG2 3D collagen microspheres was higher than that in HepG2 two-dimensional culture (P < 0.05). (4) The expression levels of albumin and CYP3A4 protein in HepaRG 3D collagen microspheres were higher than those of HepG2 3D collagen microspheres, ordinary microspheres, and two-dimensional culture (P < 0.05). (5) These results indicated the high-level expression of hepatocyte functions in 3D collagen HepaRG microsphere, which could be taken as a reference in drug metabolism evaluation in vitro and tissue engineering application.

Cytokine Expression and Cellular Phenotype in Skin Lesions of Borderline Lepromatous Leprosy during Multiple Drug Therapy / 대한피부과학회지

BACKGROUND: Distinct patterns of T cell cytokine production have been shown to influence the outcome of infection. There will be plethora of dynamic changes of T cells and their cytokine production after starting drug therapy in leprosy skin lesions. OBJECTIVE: To determine dynamics of cytokine expression, T cell and macrophage populations in the lesions taken serially in early part of World Health Organization-Multiple Drug Therapy in BL patients. METHODS: Cytokine mRNA expression of TNF-alpha, IFN-gamma, IL-4 and IL-10 in BL skin lesion was detected by RT-PCR analysis. To determine cellular phenotypes of infiltrated cells, immunohistochemical staining method was performed using antibodies to CD4, CD8, CD56, CD57, CD 68, gammadelta-TCR , and S-100 protein. RESULTS: TNF-alpha mRNA, initially showed no consistent change, increased 6 months after MDT. IFN-gamma and IL-10 mRNA showed decreasing tendency initially, but failed to show any consistent increase or decrease after 6 months. IL-4 mRNA was not detected in our patients. In reactional states, mRNA expression of TNF-alpha, IFN-gamma, IL-10 was increased in 2 patients but decreased in 1 patient compared to baseline. Ratio of TNF-alpha positive cells decreased during MDT compared to baseline(p<0.05), but ratio of cells expressing IFN-gamma showed no significant change after MDT. Only CD68 positive cells decreased after MDT(p<0.05). CONCLUSION: Variable treatment induced changes in cellular patterns and cytokine expression within BL lesion observed in this study suggest that complex mechanism of immune systems - including cytokine regulation and defect in macrophages - may exist and be involved in the pathogenesis of leprosy.

THE PHENOTYPE DIFFERENCE OF LYMPHOCYTES ACTIVATED BY IL-12 AND IL-2 AND ITS SIGNIFICANCE / 解放军医学杂志

To investigate the phenotype characteristics of peripheral blood lymphocytes(PBL)grown in the presence of IL 12 and IL 2. CD2,CD3,CD8,CD28, CD19 and NK were detected using immunofluorescence and flow cytometry. The phenotype of PBL showed significant changes after being cultured 72h with IL 12 and IL 2 respectively.IL 12 mainly increased CD2( P