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Objective To investigate the effects of granulocyte macrophage colony-stimulating factor (GM-CSF) combined with interleukin-12 (IL-12) genes on apoptosis of hepatoma cells.Methods The hepatoma cell lines were cultured in vitro and were divided into four groups: GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group,negative control group (empty load group),respectively.The PIB-CMV3-GM-CSF and PIB-CMV3-IL-12 eukayotic expression vector was built,and 36 h after transfection,fluorescence microscope was used to detect the transfection effect;the expression level of IL-12,GM-CSF,p53,p38 and C-JUN mRNA were detected by RT-PCR,and Western blot was used to examine the expression level of IL-12,GM-CSF,p53,p38 and C-JUN protein.In addition,the flow cytometry was applied to detect cell apoptosis.Results Through fluorescence microscope,green fluorescence was observed in cells of GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group,indicating that the plasmid has successfully transferred into cells.In addition,the expression of p53mRNA in empty load group,GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group were 1.2±0.10,4.3±0.98,4.2±0.34,9.2±0.87,and the protein expression were 1.0±0.10,3.6±0.34,3.8±0.30,5.0±0.60.Compared with the empty load group,the expression level of p53 mRNA and protein were significantly increased in the three plasmid transfection groups (P<0.01).The expression of p53 mRNA and protein were significantly increased in co-transfection group than GM-CSF group and IL-12 group (P<0.01),while in the comparison with GM-CSF transfection group and IL-12 transfection group,the expression level of p53mRNA and protein in the co-transfection group could be improved to a higher degree(P<0.01).Meanwhile,p38 C-JUN mRNA expression levels in empty load group,GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group were as follows: 7.5± 0.9,3.5±0.45,3.7±0.25,1.0±0.11,while p38protein expression levels were 10.1±1.03,6.1± 0.67,7.1 ± 0.61,1.0 ± 0.12,respectively,C-JUN mRNA expression levels were 11.2 ± 1.20,4.1 ± 0.19,3.3 ± 0.30,1.0 ± 0.01,separately,C-JUN protein expression levels were 2.25 ± 0.2,1.8 ± 0.13,1.4 ± 0.12,1.0 ± 0.09.P38, C-JUN mRNA and protein levels were significantly reduced in the three plasmid transfection groups compared with the empty load group (P<0.01).The expression of p38,C-JUN mRNA and protein were reduced to a lower degree in co-transfection group than in GM-CSF transfection group and IL-12 transfection group (P<0.01).Flow cytometer showed that the hepatoma cell apoptosis rate of the empty load group,GM-CSF transfection group,IL-12 transfection group,co-transfection group were (3.43±0.9)%,(5.87±1.02)%,(7.32±1.1)%,(17.47±2.11)%,the rates of the three plasmid transfection groups were significantly higher than that of the empty load group (P<0.01).And the apoptosis rate was significantly increased in the co-transfected group compared with other plasmid groups (P<0.01). Conclusion The combination of GM-CSF and IL-12 could significantly accelerate the apoptosis of hepatoma cells by up-regulating the expression of p53,and down-regulating the expression of p38 and C-JUN.
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ObjectiveTo investigate the effect of decoy receptor 3 (DcR3) gene on hepatocyte apoptosis, as well as the possible mechanism of action of DcR3 in this process. MethodsThe human liver cell lines were cultured in vitro, and pEF1α-DcR3 transfection group, pEF1α-IRES transfection group, and negative control group were established. The pEF1α-IRES-DsRed-Express2-DcR3 eukaryotic expression vector was constructed and transfected into human liver cell lines for 36 hours. qRT-PCR was used to measure the mRNA expression of DcR3, Fas ligand (FasL), α-smooth muscle actin (α-SMA), and transforming growth factor-β1 (TGF-β1), Western blot was used to measure the change in the protein expression of DcR3, and flow cytometry was used to measure apoptosis. An analysis of variance was used for comparison of continuous data between groups, and the least significant difference t-test was used for comparison between any two groups. ResultsAfter human liver cell lines were transfected with pEF1α-DcR3 for 36 hours, the pEF1α-DcR3 transfection group showed significant increases in the mRNA and protein expression of DcR3 compared with the pEF1α-IRES transfection group and negative control group (F=33 1695 and 14154, all P＜0.01). Compared with the other two groups, the pEF1α-DcR3 transfection group showed significant reductions in the mRNA expression of FasL, α-SMA, and TGF-β1 (F=269 4518, 20 7904, and 80678, all P＜0.01), which suggested that DcR3 inhibited the expression of FasL, α-SMA, and TGF-β1. Compared with the pEF1α-IRES transfection group and negative control group, the pEF1α-DcR3 transfection group showed a significant reduction in apoptosis rate (F=55863, all P＜0.01). ConclusionDcR3 can inhibit hepatocyte apoptosis and downregulate the mRNA expression of FasL, α-SMA, and TGF-β1.
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Objective:To construct the human DcR3 expression vector and verify its expression in vitro. Methods: 915 bp human DcR3 gene CDS was amplified from porcine lung tissues,and was cloned into eukaryotic expression vector pEF1a-IRES-DsRed-Express2 which show red fluorescence. And then pEF1a-IRES-DsRed-Express2-DcR3 was transfected into LX-2 cells by FuGene HD. Expression of mRNA and protein lever of Human DcR3 were detected by RT-PCR and Western blot. Results:The levels of DcR3 gene transcription and translation in the hepatic stellate cells were significantly increased after transfection with pEF1a-IRES-DsRed-Ex-press2-DcR3 by RT-PCR and Western blot analysis. Conclusion: DcR3 expression vector was successfully constructed and highly expressed in LX-2 cells.
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Objective: To observe relation among sICAM-1 level in the sera of patients with chronic viral hepatic disease, viral replication and the clinical significance. Methods: The level changes of sICAM-1, IL-1,IL-8 and TNFa in the sera of patients with chronic hepatitis B(CH),chronic severe hepatitis B(CSH) and hepatocirrhosis (HC) were detected by ELISA. Results: The levels of sICAM-1,IL-1,IL-8 and TNFa in sera of the patients with chronic viral hepatic disease were significantly higher than those of healthy control group(P<0.01). The bilirubin level in the sera of patients with chronic viral hepatic disease of different clinical types positively correlated with sICAM-1 and cytokine activities. The sICAM-1 level in the sera of HBV-DNA or HBeAg positive patients was significantly higher than those of negative patients(P<0.05,P<0.01). Conclusion: The sICAM-1 level and activities of cytokine IL-1,IL-8 and TNFa as well as the bilirubin level in the sera of the patients can reflect the necrosis degree of hepatocytes, and the sICAM-1 level and activities of IL-1,IL-8 and TNFa were related to state of HBV carrier or the active degree of HBV in patients with hepatic disease.
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Objective To investigate the effects of oxymatrine on liver fibrosis and its mechanism. Methods The rat model of liver fibrosis induced by CCl 4 was set up. The effect of oxymatrine anti-liver fibrosis were evaluated by comparing liver weight index, serum/tissue biochemical indices and the change of tissue pathology before and after oxymatrine intervention. The levels of serum HA,PⅢP,LN and Ⅳ-C were also measured before and after oxymatrine treatment. Results In CCL4-induced liver fibrosis model rats, the levels of serum HA,PⅢP,LN and Ⅳ-C significantly decreased(P
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Objective:To observe clinical significance between changes of OPN,IL-18 levels and relation ship with hepatic function in patients with chronic hepatic disease infected by HBV.Methods:The levels of OPN,IL-18 in peripheral blood of 102 patients with chronic hepatitis B,34 patients with hepatitis B-related cirrhosis,95 HBV carriers and 20 healthy people as the control group were respectively detected with ELISA.Results:The levels of OPN and IL-18 in chronic hepatitis B and hepatitis B-related cirrhosis groups were significantly increased compared with that of health control group(P
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Abstract Objective: To observe relationship among sICAM-1、TGF-?1, and cellular factor level changes in the sera of patients with chronic viral hepatic disease, viral replication and clinical significance.Methods;The sICAM-1 .TGF-?1, ,IL-1,IL-8 and TNFa level changes in the sera of patients with chronic hepatitis B(CH), chronic severe hepatitis B(CSH) and hepato-cirrhosis(HC) were detected by FJ-ISA. Re-sults: The sICAM-1, IL-1, IL-8 and TNFa levels in sera of patients with chronic viral hepatic disease were significantly higher than those of healthy group( P