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OBJECTIVE To study the regulator y mech anism of glaucocalyxin A （GLA） on autophagy and apoptosis of HCCLM3 hepatocellular carcinoma cells. METHODS HCCLM3 cells were taken ，and control group ，GLA 2.5 μg/mL group，GLA 5 μg/mL group and GLA 10 μg/mL group were mainly set according to different experimental purposes. In control group，only complete medium was added ；in each administration group ，complete medium containing the corresponding final concentration of GLA was added. Cell cycle distribution and apoptosis were detected by flow cytometry ；mitochondrial morphology and autophagy were observed by transmission electron microscope （only control group ，GLA 5 μg/mL group）；JC-1 staining and fluorescence inverted microscope were used to observe and detect the mitochondrial membrane potential of the cells ；Western blot assay was used to detect the protein expression of Bcl- 2， Bax， Beclin1 and cleaved caspase- 3 proteins in the cells ； the co-immunoprecipitation method was used to detect the binding and dissociation of Bcl- 2 and Beclin 1（only GLA 5 μg/mL group， GLA 10 μg/mL group）. RESULTS Compared with control group ，GLA 5 μg/mL and GLA 10 μg/mL could induce a significant arrest of the cell cycle in the G 2-M phase for HCCLM 3，a significant decrease in mitochondrial membrane potential ，an increase in apoptosis as well as significant promotion of the protein expression of Bax ，cleaved caspase- 3 and Beclin 1，and significant inhibition of the protein expression of Bcl- 2（P＜0.01）. GLA 5 μg/mL also significantly changed mitochondrial morphology and increased autophagosomes. The results of co-immunoprecipitation showed that compared with GLA 5 μg/mL，GLA 10 μg/mL could enhance the binding of Bcl- 2 and Beclin 1. CONCLUSIONS GLA can regulate the autophagy and apoptosis of HCCLM 3 cells by Bcl-2/Beclin1 target. The effect is closely related to the dose of GLA.
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Objective To investigate the effects of glaucocalyxin A ( GLA) on the expression of signal transduction and activator of transcription 3 (STAT3) signaling pathway in HCCLM3 cells for better understand-ing the role of GLA in tumor metastasis. Methods HCCLM3 cells were stimulated by different concentrations (0. 1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml and 1000 ng/ml) of GLA for 48 h. Expression of IL-6, STAT3/pSTAT3Tyr705 and MMP9 at mRNA and protein levels were detected by qRT-PCR and Western blot, respectively. Transwell invasion and migration assays were performed to analyze cell invasion and migration. ELISA was used to measure the concentrations of IL-6 in culture media. Results Expression of IL-6 at mRNA and protein lev-els, STAT3Tyr705 phosphorylation, and the migration and invasion of HCCLM3 cells were significantly enhanced after stimulation of HCCLM3 cells with 0. 1 ng/ml of GLA, but inhibited by GLA at the concentration of 1000 ng/ml. Moreover, more IL-6 was secreted out of the HCCLM3 cells treated with 0. 1 ng/ml of GLA. Conclu-sions GLA might affect the metastasis of HCCLM3 cells through regulating the expression of IL-6 and the phos-phorylation of STAT3Tyr705. These regulatory effects were closely related to the concentration of GLA.
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0.05). The cells on Ti1 and Ti2 were attached well till confluent.Conclusion:The neotype titanium alloys are biocompatible.
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Objective:To study the sensitization of two newly developed titanium alloys, TLE and TLM.Methods:According to ISO 10993-10:1995 standard,maximization test was conducted in guinea pig.The skin sensitization reactions,including erythema and oedema, induced by TLE, TLM, normal saline and 2,4-dinitrochlorobenzene were observed and scored respectively at 24,48 and 72 h exposure of the infusion of the materials. The allergenic rates and mean response score were calculated.Results:The allergenic rates and score of skin reaction of TLE and TLM were 0/15 and 0,those of normal saline 0/15 and 0,those of 2,4-dinitrochlo-robenzene 15/15 and 5,respectively.Conclusion: TLE and TLM both are not of sensitization.