Objective:To observe the effects of Chaihu Jiedu decoction on human hepatic stellate LX-2 cells,and to explore the potential molecular mechanisms.Methods:The wistar rats were divided into the experimental group and the control group,respectively with Chaihu Jiedudecoction and saline lavage,then centrifugal and get the drug-containing serum and control serum.At the same time,trsuscitate cells.When we got the expected number of cells,we divided into the experimental group and the control group.The human hepatic stellate cells (LX-2) were with the drug serum for 24 h,36 h,48 h,72 h.Then the cell proliferation inhibition rate was measured by CCK-8,the apoptosis were detected by flow cytometry (Annexin V-FITC,PI staining method).Results:ChaihuJiedu decoction could inhibit proliferation of LX-2 cells 24 hours after dosing.The in hibition rate in 36 h,48 h,72 h were 0.37 ％,0.46 ％,0.44 ％ respectively.It could prevent LX-2 cells into proliferation and induce the apoptosis of LX-2 cells.The apoptosis rates in 48 h,72 h were (9.80±0.95)％,(36.40± 5.09)％ respectively,and there are difference of statistical significance(P＜0.05).Conclusion:Chaihu Jiedu decoction can inhibit the proliferation of LX-2 cells proliferation,and induce apoptosis,so as to interfere with course of the liver fibrosis.

Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release (IICR) via IP(3)R(1), but the mechanism remains largely unexplored. Using extracellular ATP to induce intracellular calcium transient as an IICR model, Ca(2+) image, pull down assay, and Western blotting experiments were carried out in the present study. We found that extracellular ATP induced calcium transient via IP(3)Rs (IICR) and the IICR were markedly decreased in ERp44 overexpressed Hela cells. The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331-377) mutants of ERp44 on IICR were significantly decreased compared with ERp44. However, the binding capacity of ERp44 to L3V domain of IP(3)R(1) (1L3V) was enhanced by ERp44 C160S/C212S mutation. Taken together, these results suggest that the mutants of ERp44, C160/C212, can more tightly bind to IP(3)R(1) but exhibit a weak inhibition of IP(3)R(1) channel activity in Hela cells.
Adenosine Triphosphate ; pharmacology ; Amino Acid Substitution ; Biological Transport ; drug effects ; physiology ; Blotting, Western ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; physiology ; HeLa Cells ; Humans ; Immunoprecipitation ; Inositol 1,4,5-Trisphosphate ; metabolism ; Inositol 1,4,5-Trisphosphate Receptors ; physiology ; Membrane Potentials ; drug effects ; physiology ; Membrane Proteins ; genetics ; metabolism ; Microscopy, Confocal ; Molecular Chaperones ; genetics ; metabolism ; Mutation ; Plasmids ; Transfection

Objective:To study the characteristics of macrophage lineage model polarized to adapt to the tumor micro-environment (TME) for further research on the plasticity of macrophages in TME.Methods:Bone marrow cells from transgenic Foxn1 nu.B6-CAG-EGFP/SU mice were induced by colony-stimulating factor 1 (CSF-1), IFN-γ+ LPS and IL-4 to differentiate into M0, M1 and M2 macrophages, respectively. Green fluorescent protein (GFP) was observed under inverted fluorescence microscope. Immunocytochemical staining was used to detect the marker and polarization-related proteins of macrophages. Moreover, the macrophages were co-cultured with human glioma stem cell SU3 for further analysis. Results:The bone marrow-derived M0, M1 and M2 macrophages all showed strong green fluorescence under inverted fluorescence microscope. The inherent plasticity of the macrophages could be observed under ordinary microscope with Wright-Giemsa staining. Immunocytochemical staining showed that CD11C and CD206 markers were observed on M0, M1 and M2 macrophages, while CD68 was only expressed on M1 macrophages. Moreover, the staining was strongly positive for CSF-1 and CSF-1R on M0, M1 and M2 macrophages. Green fluorescent cell infiltration and phagocytic reaction were observed in the co-cultured stem cell spheres.Conclusions:The bone marrow-derived macrophage lineage including M0, M1 and M2 subtypes with the inherent plasticity was successfully prepared using transgenic nude mice expressing GFP. The three subtypes expressed the common marker and polarization-related proteins, and had the phagocytic activity, suggesting that they could be used to study the interaction between tumor cells and macrophages, especially in tracer studies.

Objective:To establish a three-dimensional spheroid expansion model of human glioma stem cells with spontaneous sphere forming and multilineage differentiation potential in vitro and investigated the contents of the proteins and lipids and their secondary components, so as to lay a foundation for further study of sphere metabolism. Methods:Human glioma stem cells GSC23 and SU3 were cultured in serum-free stem cell culture medium, respectively, and the cell spheres were harvested for about 2-3 weeks. After fixation in paraformaldehyde solution, dehydration, paraffin embedding, and sectioning, glioma-associated marker proteins were detected by immunohistochemical staining, and the protein and lipid and their secondary components contents in the spheroid tissues were analyzed by Raman imaging. One-way ANOVA was used to compare the protein, lipid and phenylalanine contents in the large sphere, medium sphere and small sphere groups.Results:Both stem cells were able to form stem cell expansion spheres resembling solid tumors within the culture dish. Immunohistochemical staining showed that the regular marker proteins of glioblastoma multiforme, CD133, nestin, epidermal growth factor receptor (EGFR), S100, Olig2, p53, Ki-67, glial fibrillary acidic protein (GFAP), vimentin, CXC chemokine receptor 4 (CXCR4), and CD34, were all expressed. Raman imaging revealed that the constructed expanded spheres of human glioma stem cells contained protein (2 930, 1 685 and 1 586 cm -1), lipid (2 845 and 1 444 cm -1), phenylalanine (1 003 cm -1) amide Ⅲ (1 250 cm -1), while there were no significant differences of protein, lipid and phenylalanine contents among the large sphere, medium sphere and small sphere groups ( P>0.05). Conclusions:We have successfully established an expanded spheroid model of human glioma stem cells in vitro, which not only exhibits the topographical characteristics and unlimited expansion ability of three-dimensional solid tumors, but also has the ability to stably store metabolically obligatory energy sources of tumor cells such as proteins and lipids, and is expected to serve as a promising tool for human glioma research in vitro.

Objective:To establish a three-dimensional spheroid expansion model of human glioma stem cells with spontaneous sphere forming and multilineage differentiation potential in vitro and investigated the contents of the proteins and lipids and their secondary components, so as to lay a foundation for further study of sphere metabolism. Methods:Human glioma stem cells GSC23 and SU3 were cultured in serum-free stem cell culture medium, respectively, and the cell spheres were harvested for about 2-3 weeks. After fixation in paraformaldehyde solution, dehydration, paraffin embedding, and sectioning, glioma-associated marker proteins were detected by immunohistochemical staining, and the protein and lipid and their secondary components contents in the spheroid tissues were analyzed by Raman imaging. One-way ANOVA was used to compare the protein, lipid and phenylalanine contents in the large sphere, medium sphere and small sphere groups.Results:Both stem cells were able to form stem cell expansion spheres resembling solid tumors within the culture dish. Immunohistochemical staining showed that the regular marker proteins of glioblastoma multiforme, CD133, nestin, epidermal growth factor receptor (EGFR), S100, Olig2, p53, Ki-67, glial fibrillary acidic protein (GFAP), vimentin, CXC chemokine receptor 4 (CXCR4), and CD34, were all expressed. Raman imaging revealed that the constructed expanded spheres of human glioma stem cells contained protein (2 930, 1 685 and 1 586 cm -1), lipid (2 845 and 1 444 cm -1), phenylalanine (1 003 cm -1) amide Ⅲ (1 250 cm -1), while there were no significant differences of protein, lipid and phenylalanine contents among the large sphere, medium sphere and small sphere groups ( P>0.05). Conclusions:We have successfully established an expanded spheroid model of human glioma stem cells in vitro, which not only exhibits the topographical characteristics and unlimited expansion ability of three-dimensional solid tumors, but also has the ability to stably store metabolically obligatory energy sources of tumor cells such as proteins and lipids, and is expected to serve as a promising tool for human glioma research in vitro.

Ambystoma mexicanum/immunology* ; Amputation ; Animals ; Biomarkers/metabolism* ; Blastomeres/immunology* ; Cell Lineage/immunology* ; Connective Tissue Cells/immunology* ; Epithelial Cells/immunology* ; Forelimb ; Gene Expression ; High-Throughput Nucleotide Sequencing ; Humans ; Immunity ; Peroxiredoxins/immunology* ; Regeneration/immunology* ; Regenerative Medicine/methods* ; Single-Cell Analysis/methods*