Objective To explore the role of HIV-1 tat gene variations in AIDS dementia complex （ADC） pathogenesis. Methods HIV-1 tat genes derived from peripheral spleen and central basal ganglia of an AIDS patient with ADC an...Objective To explore the role of HIV-1 tat gene variations in AIDS dementia complex （ADC） pathogenesis. Methods HIV-1 tat genes derived from peripheral spleen and central basal ganglia of an AIDS patient with ADC and an AIDS patient without ADC were cloned for sequence analysis. HIV-1 tat gene sequence alignment was performed by using CLUSTAL W and the phylogentic analysis was conducted by using Neighbor-joining with MEGA4 software. All tat genes were used to construct recombinant retroviral expressing vector MSCV-IRES-GFP/tat. The MSCV-IRES-GFP/tat was cotransfected into 293T cells with pCMV-VSV-G and pUMVC vectors to assemble the recombinant retrovirus. After infection of gliomas U87 cells with equal amount of the recombinant retrovirus, TNF-α, and IL-1β concentrations in the supernatant of U87 cells were determined with ELISA. Results HIV-1 tat genes derived from peripheral spleen and central basal ganglia of the AIDS patient with ADC and the other one without ADC exhibited genetic variations. Tat variations and amino acid mutation sites existed mainly at Tat protein core functional area （38-47aa）. All Tat proteins could induce ug7 cells to produce TNF-α and IL-1β, but the level of IL-1β production was different among Tat proteins derived from the ADC patient＇s spleen, basal ganglia, and the non-ADC patient＇s spleen. The level of Tat proteins derived from the ADC patient＇s spleen, basal ganglia, and the non-ADC patient＇s spleen were obviously higher than that from the non-ADC patient＇s basal ganglia. Conclusion Tat protein core functional area （38-47aa） may serve as the key area of enhancing the secretion of IL-1β. This may be related with the neurotoxicity of HIV-1 Tat.展开更多
Objective： To provide the experimental basis for further studying the molecular transformation mechanism of Hepatitis B virus （HBV） X protein （HBx） on hepatocellular carcinoma. Methods： Reconstructed plasmid pcD...Objective： To provide the experimental basis for further studying the molecular transformation mechanism of Hepatitis B virus （HBV） X protein （HBx） on hepatocellular carcinoma. Methods： Reconstructed plasmid pcDNA3.1（＋）-HBx was transfected into THP-1 macrophages. Expression of HBx was assayed in macrophages lysate by Western-blotting, and TNF-α and IL-1β contents were detected respectively by ELISA. All the data were analyzed by SPSS13.0. Results： In THP-lmacrophages, the pcDNA3.1（＋）-HBx plasmid expressed HBx with a molecular weight of about 17 KDa demonstrated by Western-blotting. The secreted TNF-α and IL-1β from macrophages were determined by ELISA, the results from analysis of all groups showed as following： control group was different from LPS group and pcDNA3.1（＋） group （P〈0.01）, and so was pcDNA3.1（＋）-HBx group; but there was no obvious difference between pcDNA3.1（＋） group and LPS group （P〉0.05）, all of which indicated that transient overexpression of HBx enhanced LPS-induced production of TNF-α and IL-1β by macrophages. Conclusion： Transient overexpression of HBx up-regulates LPS-induced TNF-t~ and IL-113 secretion of macrophages.展开更多
基金supported by the Science&Technology Development Program of Shandong Province(Grant No.2007GG30002003)
文摘Objective To explore the role of HIV-1 tat gene variations in AIDS dementia complex （ADC） pathogenesis. Methods HIV-1 tat genes derived from peripheral spleen and central basal ganglia of an AIDS patient with ADC and an AIDS patient without ADC were cloned for sequence analysis. HIV-1 tat gene sequence alignment was performed by using CLUSTAL W and the phylogentic analysis was conducted by using Neighbor-joining with MEGA4 software. All tat genes were used to construct recombinant retroviral expressing vector MSCV-IRES-GFP/tat. The MSCV-IRES-GFP/tat was cotransfected into 293T cells with pCMV-VSV-G and pUMVC vectors to assemble the recombinant retrovirus. After infection of gliomas U87 cells with equal amount of the recombinant retrovirus, TNF-α, and IL-1β concentrations in the supernatant of U87 cells were determined with ELISA. Results HIV-1 tat genes derived from peripheral spleen and central basal ganglia of the AIDS patient with ADC and the other one without ADC exhibited genetic variations. Tat variations and amino acid mutation sites existed mainly at Tat protein core functional area （38-47aa）. All Tat proteins could induce ug7 cells to produce TNF-α and IL-1β, but the level of IL-1β production was different among Tat proteins derived from the ADC patient＇s spleen, basal ganglia, and the non-ADC patient＇s spleen. The level of Tat proteins derived from the ADC patient＇s spleen, basal ganglia, and the non-ADC patient＇s spleen were obviously higher than that from the non-ADC patient＇s basal ganglia. Conclusion Tat protein core functional area （38-47aa） may serve as the key area of enhancing the secretion of IL-1β. This may be related with the neurotoxicity of HIV-1 Tat.
基金This work was supported by Natural Science Foundationof Hunan Province (No. 06JJ4108)the Hygienic Committee of Hunan Province (No. B2005-084).
文摘Objective： To provide the experimental basis for further studying the molecular transformation mechanism of Hepatitis B virus （HBV） X protein （HBx） on hepatocellular carcinoma. Methods： Reconstructed plasmid pcDNA3.1（＋）-HBx was transfected into THP-1 macrophages. Expression of HBx was assayed in macrophages lysate by Western-blotting, and TNF-α and IL-1β contents were detected respectively by ELISA. All the data were analyzed by SPSS13.0. Results： In THP-lmacrophages, the pcDNA3.1（＋）-HBx plasmid expressed HBx with a molecular weight of about 17 KDa demonstrated by Western-blotting. The secreted TNF-α and IL-1β from macrophages were determined by ELISA, the results from analysis of all groups showed as following： control group was different from LPS group and pcDNA3.1（＋） group （P〈0.01）, and so was pcDNA3.1（＋）-HBx group; but there was no obvious difference between pcDNA3.1（＋） group and LPS group （P〉0.05）, all of which indicated that transient overexpression of HBx enhanced LPS-induced production of TNF-α and IL-1β by macrophages. Conclusion： Transient overexpression of HBx up-regulates LPS-induced TNF-t~ and IL-113 secretion of macrophages.
