ABSTRACT
Objective To identify the host-cell death pathways (apoptosis, autophagy or necrosis) in L929 cells at the time point of 48 hours post infection (h.p.i.) with Chlamydia muridarum.Methods L929 cells were infected with Chlamydia muridarum at a multiplicity of infection (MOI) of 0.85 for 48 hours.Nuclear fragmentation was observed under fluorescence microscopy following staining L929 cells with DAPI (4′,6-diamidino-2-phenylindole).L929 cells were stained with propidium iodide (PI) plus Annexin Ⅴ and then analyzed by fluorescence-activated cell sorting (FACS) to clarify whether apoptosis or necrosis occurred after Chlamydia muridarum infection.L929 cells were transiently transfected with GFP-LC3 and observed under fluorescent microscopy to analyze cell autophagy.Western blot assay was performed to detect LC3 protein for further analysis of autophagy.Results Apoptosis was not induced in L929 cells by Chlamydia muridarum infection at 48 h.p.i.as no significant nuclear fragmentation was observed.Results of FACS showed that most cells died due to necrosis.Moreover, fluorescent dots of GFP-LC3 formed after infecting transfected L929 cells with Chlamydia muridarum.An increased ratio of LC3Ⅰ to LC3Ⅱ in the L929 cells infected with Chlamydia muridarum was detected by Western blot assay, indicating that autophagy occurred during Chlamydia muridarum infection.Conclusion Necrosis and autophagy rather than apoptosis are induced in L929 cells 48 hours after infection with Chlamydia muridarum.
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Collagen is an extracellular matrix protein with great importance in biomedical application. The search for collagen from various sources is intensified especially from marine source. This study was carried out to extract collagen from a Malaysian local fresh water fish, Clarias batrachus and characterized its biomedical potential In vitro. Collagen was extracted from C. batrachus skin using acetic acid method and identified using SDS-PAGE. MTT assay was performed to determine the effect of coated collagen on cell adhesion and proliferation of L929 skin fibroblast cells. Additionally, scratch assay was performed to examine the effect of collagen coating on fibroblast cell migration. Result showed that collagen extracted from C. batrachus was made up of collagen type I, which consists of two α chains (α-1 and α-2) and β chain. At 100 μg/cm2 density, collagen coating significantly increased fibroblast cell adhesion, proliferation and migration compared to negative control (p< 0.05). As a conclusion, collagen extracted from C. batrachus increased cell adhesion, proliferation and migration of fibroblast cells and has potential to be used as an alternative source of collagen.
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Objective To observe the influence of peroxisome proliferator activated receptor-γligand (PPAR-γ, pioglatazone) on expression of PAI-1 and TGF-β mRNA and proliferation in fibroblast cells before and after X-ray radiation, and to study the effect of PPAR-γon normal cells during radiation induced fibrosis process. Methods RT-PCR method was used to measure PPAR-γgene expression in L929 cells.After X-ray irradiation of 10 Gy,4 Gy or 2 Gy, the expressions of PAI-1 and TGF-β mRNA in mouse lung fibroblast cells (L929) were measured using RT-PCR. After X-ray irradiation and pioglatazone treatment,the influence of pioglatazone on PAI-1 and TGF-β was measured using RT-PCR method. MTT method was used to test cell proliferation after the treatment of irradiation and pioglatazone. Results PPAR-γ mRNA expression was observed in L929 cells. Expression of PAI-1 and TGF-β mRNA reached the highest level 483.40,P =0. 090) ). At 48 h after the treatment of pioglatazone and 10 Gy radiation, pioglatazone decreased 0. 36, 0. 34 and 0. 32( F = 3.90, P = 0. 040) ). The inhibitory effect was significantly increased when L9292. 50,P =0. 005)). Conclusions X-ray irradiation can increase the expression of PAI-1 and TGF-β in L929 cells. Pioglatazone can decrease the expression of radiation-induced PAI-1 and TGF-β, and restrain the fibroblast proliferation.
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AIM To investigate the nongenomic effects of steroids on glycine uptake in L 929 cells. METHODS L 929 cells were incubated with labeled glycine, steroids, and/or other reagents. With liquid scintillation technique, the labeled glycine in L 929 was measured. RESULTS Steroids could rapidly inhibit the glycine uptake. Action intensity of steroids was different. Effects of CORT and ALD were dose-dependent. There was no difference in effects between corticosterone 21-sulfate and B-BSA. Inhibitor for cytoplasm receptor of glucocorticoids could partially block the effect of CORT. Extracelullar Ca 2+ could influence the effect of CORT. CONCLUSION Effects of steroids on glycine uptake in L 929 cells are nongenomic. Steroids may take effect through membrane receptors. The receptors of CORT in membrane are similar to those of glucocorticoids in cytoplasm.