AIM： To generate and characcerize the synthetic transoiptional control units for transcriptional targeting of the liver, thereby compensating for the lack of specificity of currently available gene therapeutic vector...AIM： To generate and characcerize the synthetic transoiptional control units for transcriptional targeting of the liver, thereby compensating for the lack of specificity of currently available gene therapeutic vector systems. METHODS： Synthetic transcriptional control unit constructs were generated and analyzed for transcriptional activities in different cell types by FACS quantification, semi-quantitative RT-PCR, and Western blotting. RESULTS： A new bifunctionally-enhanced green fluorescent protein （EGFP）/neor fusion gene cassette was generated, and could flexibly be used both for transcript quantification and for selection of stable cell clones. Then, numerous synthetic transcriptional control units consisting of a minimal promoter linked to “naturally” derived composite enhancer elements from liver-specific expressed genes or binding sites of liver-specific transcription factors were inserted upstream of this reporter cassette. Following liposome-mediated transfection, EGFP reporter protein quantification by FACS analysis identified constructs encoding multimerized composite elements of the apolipoprotein B100 （APOB） promoter or the ornithin transcarbamoylase （OTC） enhancer to exhibit maximum transcriptional activities in liver originating cell lines, but only background levels in non-liver originating cell lines. In contrast, constructs encoding only singular binding sites of liver-specific transcription factors, namely hepatocyte nuclear factor （HNF）, HNF3, HNF4, HNF5, or CAAT/enhancer binding protein （C/EBP） only achieved background levels of EGFP expression. Finally, both semi-quantitative RT-PCR and Western blotting analysis of Hep3B cells demonstrated maximum transcriptional activities for a multimeric 4xAPOB cassette construct, which fully complied with the dataobtained by initial FACS analysis CONCLUSION： Synthetic transcriptional control unit constructs not only exhibit a superb degree of structural compactness, but also provide new means for liver-directed expression of therapeutic genes.展开更多
BACKGROUND: Oxidative stress plays a pathogenetic role in initiation and progression of hepatic damage caused by alcohol. Recently, antioxidants have received considerable attention. Green tea polyphenols have both an...BACKGROUND: Oxidative stress plays a pathogenetic role in initiation and progression of hepatic damage caused by alcohol. Recently, antioxidants have received considerable attention. Green tea polyphenols have both antioxidant and antiinflammatory properties. This study was designed to evaluate the effect of tea polyphenol ( TP) on alcohol-induced liver injury in rats. METHODS: The rats were divided randomly into 3 groups: group A gastrically infused with alcohol for 12 weeks, group B fed with alcohol plus TP (250 mg/kg·d) simultaneously , and group C (control group) gastrically infused with normal saline. At the end of 12 weeks, the rats were sacrificed. The liver specimen of each rat was taken for his-tological examination. All data were statistically analyzed in quantum and semi-quantum. Gene expression of cytokines of each group was determined. RESULTS: At the end of 12 weeks, hepatic injury of different degrees developed in group A and group B compared to group C. The degree of hepatic injury was attenuated in group B, with slight steatosis, liver cellular swelling in small areas; less spot and focal necrosis, no bridging necrosis, less mega-bubble steatosis and less collagen deposition in contrast to group A. Gene expressions of IL-3, IL-4, IL-1R2, IL-6R, IL-7R2 were up-regulated in group B compared with group A, but those of IL-3Ra, IL-1R1 were down-regulated. Gene expressions of IL-13, IL-1R1, IL-7R2, EPO-R, LIFR were up-regulated in group A compared with group C, but those of IL-1R2, IL-5R2, CSF1, CD27, IL-6R were down-regulated. CONCLUSION: TP is able to attenuate alcoholic liver damage. Cytokine may contribute to alcoholic liver disease.展开更多
This study aimed to determine the effects of black tea polyphenols on gene expression in hepatocellular cancer cells.The total RNA from HepG_(2) hepatocellular cancer cells treated with black tea polyphenols was subje...This study aimed to determine the effects of black tea polyphenols on gene expression in hepatocellular cancer cells.The total RNA from HepG_(2) hepatocellular cancer cells treated with black tea polyphenols was subjected to Human 14K cDNA microarray analysis.Real-time PCR and Western blot analysis were conducted to verify microarray data.Black tea polyphenols treatment at the dose of 20 mg/L,40 mg/L or 80 mg/L for one to three days inhibited the growth of HepG_(2) cells in a dose and time dependent manner.A total of 48 genes showed more than two-fold change after black tea polyphenols treatment,including 17 upregulated genes and 31 downregulated genes,and they were involved in the regulation of cell growth,cell cycle,apoptosis,signaling,angiogenesis,tumor invasion and metastasis.Real-time PCR analysis of the selected genes showed that their mRNA expression changes were consistent with the microarray data.In addition,Western blot analysis of the selected genes showed that their protein expression changes were consistent with mRNA expression.In conclusion,gene expression profiles provide comprehensive molecular mechanisms by which black tea polyphenols exerts growth inhibition effects on cancer cells.The novel molecular targets identified in this study may be further exploited as therapeutic strategies for hepatocellular cancer.展开更多
基金Supported by German Research Foundation (LA 649/11-1)Fortüne-program of the Medical Faculty of the Eberhard-Karls-University Tubingen (F.128101.1) Scholarship from the Pinguin Foundation (Henkel KGaA)
文摘AIM： To generate and characcerize the synthetic transoiptional control units for transcriptional targeting of the liver, thereby compensating for the lack of specificity of currently available gene therapeutic vector systems. METHODS： Synthetic transcriptional control unit constructs were generated and analyzed for transcriptional activities in different cell types by FACS quantification, semi-quantitative RT-PCR, and Western blotting. RESULTS： A new bifunctionally-enhanced green fluorescent protein （EGFP）/neor fusion gene cassette was generated, and could flexibly be used both for transcript quantification and for selection of stable cell clones. Then, numerous synthetic transcriptional control units consisting of a minimal promoter linked to “naturally” derived composite enhancer elements from liver-specific expressed genes or binding sites of liver-specific transcription factors were inserted upstream of this reporter cassette. Following liposome-mediated transfection, EGFP reporter protein quantification by FACS analysis identified constructs encoding multimerized composite elements of the apolipoprotein B100 （APOB） promoter or the ornithin transcarbamoylase （OTC） enhancer to exhibit maximum transcriptional activities in liver originating cell lines, but only background levels in non-liver originating cell lines. In contrast, constructs encoding only singular binding sites of liver-specific transcription factors, namely hepatocyte nuclear factor （HNF）, HNF3, HNF4, HNF5, or CAAT/enhancer binding protein （C/EBP） only achieved background levels of EGFP expression. Finally, both semi-quantitative RT-PCR and Western blotting analysis of Hep3B cells demonstrated maximum transcriptional activities for a multimeric 4xAPOB cassette construct, which fully complied with the dataobtained by initial FACS analysis CONCLUSION： Synthetic transcriptional control unit constructs not only exhibit a superb degree of structural compactness, but also provide new means for liver-directed expression of therapeutic genes.
文摘BACKGROUND: Oxidative stress plays a pathogenetic role in initiation and progression of hepatic damage caused by alcohol. Recently, antioxidants have received considerable attention. Green tea polyphenols have both antioxidant and antiinflammatory properties. This study was designed to evaluate the effect of tea polyphenol ( TP) on alcohol-induced liver injury in rats. METHODS: The rats were divided randomly into 3 groups: group A gastrically infused with alcohol for 12 weeks, group B fed with alcohol plus TP (250 mg/kg·d) simultaneously , and group C (control group) gastrically infused with normal saline. At the end of 12 weeks, the rats were sacrificed. The liver specimen of each rat was taken for his-tological examination. All data were statistically analyzed in quantum and semi-quantum. Gene expression of cytokines of each group was determined. RESULTS: At the end of 12 weeks, hepatic injury of different degrees developed in group A and group B compared to group C. The degree of hepatic injury was attenuated in group B, with slight steatosis, liver cellular swelling in small areas; less spot and focal necrosis, no bridging necrosis, less mega-bubble steatosis and less collagen deposition in contrast to group A. Gene expressions of IL-3, IL-4, IL-1R2, IL-6R, IL-7R2 were up-regulated in group B compared with group A, but those of IL-3Ra, IL-1R1 were down-regulated. Gene expressions of IL-13, IL-1R1, IL-7R2, EPO-R, LIFR were up-regulated in group A compared with group C, but those of IL-1R2, IL-5R2, CSF1, CD27, IL-6R were down-regulated. CONCLUSION: TP is able to attenuate alcoholic liver damage. Cytokine may contribute to alcoholic liver disease.
基金supported by grants from the Science and Technology Foundation of Health and Family Planning Commission of Hunan Province(No.C2019048)Science and Technology Bureau of Changsha(No.ZD1702024).
文摘This study aimed to determine the effects of black tea polyphenols on gene expression in hepatocellular cancer cells.The total RNA from HepG_(2) hepatocellular cancer cells treated with black tea polyphenols was subjected to Human 14K cDNA microarray analysis.Real-time PCR and Western blot analysis were conducted to verify microarray data.Black tea polyphenols treatment at the dose of 20 mg/L,40 mg/L or 80 mg/L for one to three days inhibited the growth of HepG_(2) cells in a dose and time dependent manner.A total of 48 genes showed more than two-fold change after black tea polyphenols treatment,including 17 upregulated genes and 31 downregulated genes,and they were involved in the regulation of cell growth,cell cycle,apoptosis,signaling,angiogenesis,tumor invasion and metastasis.Real-time PCR analysis of the selected genes showed that their mRNA expression changes were consistent with the microarray data.In addition,Western blot analysis of the selected genes showed that their protein expression changes were consistent with mRNA expression.In conclusion,gene expression profiles provide comprehensive molecular mechanisms by which black tea polyphenols exerts growth inhibition effects on cancer cells.The novel molecular targets identified in this study may be further exploited as therapeutic strategies for hepatocellular cancer.
