小鼠白蛋白启动子的组织特异性功能研究

以绿色荧光蛋白(GFP)基因作为报告基因,通过对比小鼠白蛋白启动子在不同来源细胞系中启动HGFP基因的转录活性,对小鼠白蛋白启动子的组织特异性进行了研究。结果发现,小鼠白蛋白启动子在小鼠肝癌细胞系Hepa 1—6和人肝癌细胞系：HepG2均有很强的转录起始功能,荧光显微镜下可以观察到IGFP表达。Hepa 1—6细胞在转染早期的48h内,CMV的启动子和增强子序列是小鼠白蛋白启动子转录活性的4倍。G418加压筛选2周后,CMV的启动子的转录活性下降到只有小鼠白蛋白启动子活性的1／2。转染人肝癌细胞系HepG2 2周后,荧光显微镜下可以观察到GFP表达。其他的细胞如中华仓鼠卵巢细胞系CHO和人肺癌细胞系PLA 801中转染的小鼠白蛋白启动子不能启动GFP的表达,而对照CMV启动子控制下的GFP基因可在CHO和PLA 801中表达。以上结果说明,小鼠白蛋白启动子仅在肝脏来源的细胞中可以起始下游基因的转录,在其他组织来源的细胞中不能起始转录,这表明小鼠白蛋白启动子具有肝脏组织特异的转录活性,但没有种属特异性。     

Tissue-specific and insulin-dependent expression of a pancreatic amylase gene in transgenic mice.

The regulatory properties of mouse pancreatic amylase genes include exclusive expression in the acinar cells of the pancreas and dependence on insulin and glucocorticoids for maximal expression. We have characterized a murine pancreatic amylase gene, Amy-2.2y, whose promoter sequence is 30% divergent from those of previously sequenced amylase genes. To localize sequences required for tissue-specific and hormone-dependent activation, we established two lines of transgenic mice. The first line contained a single copy of the complete Amy-2.2y gene as well as 9 kilobases of 5'-flanking sequence and 5 kilobases of 3'-flanking sequence. The second line carried a minigene which included 208 base pairs of 5'-flanking sequence and 300 base pairs of 3'-flanking sequence. In both lines the transgene was expressed at high levels exclusively in the pancreas. Both constructs were dependent on insulin and induced by dexamethasone. Thus, the transferred genes contained the sequences required for tissue-specific and hormonally regulated expression.     

Hepatitis C virus is restricted at both entry and replication in mouse hepatocytes

Human hepatitis C virus (HCV) does not replicate in mouse hepatocytes. The underlying mechanisms are largely unknown. In this study, we took advantage of a series of direct and unique molecular strategies and dissected the HCV life cycle in human hepatoma Huh7.5 cells and mouse hepatoma Hepa1-6 cells. We showed that HCV entry was restricted in Hepa1-6 cells and was not rescued by expression of human HCV receptors. We also showed that HCV RNA replication was impaired in Hepa1-6 cells. In contrast, we showed that the HCV IRES translation activity and HCV production in Hepa1-6 cells were either comparable to, or even slightly higher than those in Huh7.5 cells. Thus, we conclude that entry and RNA replication are the two major HCV restrictions in mouse cells. These studies provide new insights into HCV interaction with mouse cells and new clues for formulating strategies for development of HCV mouse models.     

α-SMA与肿瘤发展相关性的研究

以小鼠的肝、肺组织的正常细胞与肝肿瘤细胞（Hepa1-6）、肺肿瘤细胞（LLC）为研究对象,通过CTFM（cell traction force microscopy）法测定了4组细胞系的牵引力;用荧光抗体染色技术比较了小鼠细胞的α-SMA蛋白在癌变前后的变化。实验发现：与小鼠正常细胞相比,在α-SMA的表达水平上,Hepa1-6细胞α-SMA的平均光密度减小了47.9%,LLC细胞下降了52.3%;而Hepa1-6细胞牵引力的均方根值减小了53.4%,LLC细胞减小了49.7%。这说明α-SMA的表达与牵引力的变化以及细胞癌变的过程是密切相关的。     

Caveolin-1真核表达载体的构建及其Hepa1-6细胞中的表达

Caveolin-1在不同肿瘤中发挥作用不同,既发挥抑癌基因样作用又发挥癌基因样作用.旨在分析caveolin-1 在小鼠肝癌细胞系中的表达情况及建立稳定表达外源caveolin-1的Hepa1-6细胞.利用RT-PCR和Western-blot方法检测caveolin-1在小鼠肝癌H22、Hea-F和Hepa1-6细胞中的表达;通过分子克隆构建小鼠caveolin-1 cDNA真核表达栽体,利用脂质体转染等方法建立稳定表达外源caveolin-1的Hepa1-6细胞株;通过RT-PCR、Western-blot、免疫细胞化学等方法鉴定其稳定表达细胞株.结果显示,caveolin-1在Hepa1-6细胞中表达呈阴性,在H22和Hca-F 中高表达;成功获得小鼠caveolin-1 cDNA真核表达载体pEGFP-N2/Cav-1,筛选并鉴定出高表达外源caveolin-1的Hepa1-6稳定细胞株C1和C4,为进一步分析caveolin-1在肝癌中所发挥的作用奠定了一定的研究基础.     

Caveolin-1 promotes the transformation and anti-apoptotic ability of mouse hepatoma cells

Caveolin-1 is a major structural protein of caveolae and plays important roles in signal transduction, cellular transformation and tumor metastasis. Our previous study demonstrated that caveolin-1 expression level was positively correlated with the invasive ability of mouse hepatoma Hepa1-6 and Hca-F cells. However, the role of caveolin-1 in cellular transformation and apoptosis remains undetermined. We found that exogenous expression of caveolin-1 in Hepa1-6 cells enhanced cell transformation capability both in vitro and in vivo and prevented actinomycin D-induced apoptosis via the activation of survivin-mediated survival pathway. Conversely, downregulation of caveolin-1 in Hca-F cells significantly attenuated cell transformation ability in vitro and in vivo and increased cell sensitivity to actinomycin D by inhibiting survivin-mediated survival pathway. These results indicate that caveolin-1 could play an active role in mediating the transformation and survival of mouse hepatoma cells and might be a potential target for gene and antitumor drugs therapy.     

Cell-type specificity of ectonucleotidase expression and upregulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin

We report here that induction of ectoATPase by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is cell-type specific and not a generalized response to aryl hydrocarbon (Ah) receptor activation. TCDD increased [14C]-ATP and -ADP metabolism in two mouse hepatoma lines, Hepa1c1c7 and Hepa1-6 cells, but not in human hepatoma HepG2 or HuH-7 cells, human umbilical vein endothelial cells (HUVEC), chick hepatoma (LMH) cells, or chick primary hepatocytes or cardiac myocytes, even though all of those cell types were Ah receptor-responsive, as evidenced by cytochrome P4501A induction. To determine whether the differences in ectonucleotidase responsiveness to TCDD might be related to differences in cell-type ectonucleotidase expression, ATP and ADP metabolite patterns, the products of several classes of ectonucleotidases including ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), ectophosphodiesterase/pyrophosphatases (E-NPP enzymes) and ectoalkaline phosphatase activities were examined. Those patterns, together with results of enzyme assays, Western blotting, or semiquantitative RT-PCR show that NTPDase2 is the main ectonucleotidase for murine and human hepatoma cells, NTPDase3 for chick hepatocytes and LMH cells, and an E-NPP enzyme for chick cardiac myocytes. Evidence for NTPDase2 expression was lacking in all cells except the mouse and human hepatoma cells. TCDD increased expression of the NTPDase2 gene but only in the mouse and not in the human hepatoma cells. TCDD did not increase NTPDase3, NTPDase1, E-NPP, or alkaline phosphatase in any of the cell types examined. The failure of TCDD to increase ATP metabolism in HUVEC, chick LMH cells, hepatocytes, and cardiac myocytes can be attributed to their lack of NTPDase2 expression, while the increase in ATP metabolism by TCDD in the mouse but not the human hepatoma cells can be explained by differences in TCDD effects on mouse and human hepatoma NTPDase2 gene expression. In addition to characterizing effects of TCDD on ectonucleotidases, these studies reveal major differences in the complements of ectonucleotidases present in different cell types. It is likely that such differences are important for cell-specific susceptibility to extracellular nucleotide toxicity and responses to purinergic signaling.     

Interferon-stimulated gene ISG12b2 is localized to the inner mitochondrial membrane and mediates virus-induced cell death

Interferons (IFNs) are crucial for host defence against viruses. Many IFN-stimulated genes (ISGs) induced by viral infection exert antiviral effects. Microarray analysis of gene expression induced in liver tissues of mice on dengue virus (DENV) infection has led to identification of the ISG gene ISG12b2. ISG12b2 is also dramatically induced on DENV infection of Hepa 1-6 cells (mouse hepatoma cell line). Here, we performed biochemical and functional analyses of ISG12b2. We demonstrate that ISG12b2 is an inner mitochondrial membrane (IMM) protein containing a cleavable mitochondrial targeting sequence and multiple transmembrane segments. Overexpression of ISG12b2 in Hepa 1-6 induced release of cytochrome c from mitochondria, disruption of the mitochondrial membrane potential, and activation of caspase-9, caspase-3, and caspase-8. Treatment of ISG12b2-overexpressing Hepa 1-6 with inhibitors of pan-caspase, caspase-9, or caspase-3, but not caspase-8, reduced apoptotic cell death, suggesting that ISG12b2 activates the intrinsic apoptotic pathway. Of particular interest, we further demonstrated that ISG12b2 formed oligomers, and that ISG12b2 was able to mediate apoptosis through both Bax/Bak-dependent and Bax/Bak-independent pathways. Our study demonstrates that the ISG12b2 is a novel IMM protein induced by IFNs and regulates mitochondria-mediated apoptosis during viral infection.     

Regulatory factors involved in species-specific modulation of arylhydrocarbon receptor (AhR)-dependent gene expression in humans and mice

The arylhydrocarbon receptor (AhR) mediates toxicities of dioxins, including the most potent congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), by being translocated to the nucleus upon ligand-binding and inducing expression of target genes. Although the species-specific activity of the AhR is primarily attributable to species-specific AhR-ligand affinity, the precise mechanism has not been clarified. We investigated the modulation mechanisms of AhR in Hepa1c1c7 and HepG2 hepatoma cells, which were derived from high-affinity-AhR-expressing C57BL/6 mice and low-affinity-AhR-expressing humans, respectively. Although, consistent with their AhR affinities, TCDD induced a greater amount of cytochrome P450 1A1 (CYP1A1) mRNA, one of the most sensitive AhR-targets, in Hepa1c1c7 cells than in HepG2 cells immediately after exposure, both cells expressed a similar level of CYP1A1 mRNA from 4 h onward. A rapid decrease in the AhR protein after nuclear translocation in Hepa1c1c7 cells was suggested to contribute to suppression of CYP1A1 induction to the same level as in HepG2 cells. Different profiles of histone deacetylase 1 (HDAC1)-binding to the CYP1A1 promoter and histone acetylation between both cell lines and lower degradation rate of CYP1A1 mRNA in HepG2 cells were also implicated in regulating their target gene expression. These factors have been highly suggested to be involved in the species-specific modulation mechanism of AhR function.     

Alpha-syntrophin dependent expression of tubulin alpha 8 protein in hepatocytes

The scaffold protein alpha-syntrophin (SNTA) is a component of the dystrophin glycoprotein complex and has been comprehensively studied in skeletal muscle and adipocytes. SNTA is further expressed in the liver where its biological role remains unclear. Unpublished data from our group suggested that SNTA deficiency is associated with altered tubulin alpha 8 (TUBA8) levels in fat. TUBA8 is highly expressed in different cell lines including hepatoma cells, and here we analyzed whether SNTA has a role herein. In Hepa1-6 cells, TUBA8 protein levels were increased upon SNTA knock down and were reduced upon overexpression of SNTA. This regulation was not identified when analyzing mRNA expression. In the liver of SNTA-deficient mice, TUBA8 protein was higher compared to the respective wild-type controls while RNA expression was even suppressed. Using the HaloTag platform, TUBA8 was found to form a complex with SNTA in Hepa1-6 cells. In the hepatic stellate cell line LX-2, the lack or overexpression of SNTA did, however, not change TUBA8 protein expression. SNTA and TUBA8 are described to regulate cell proliferation. Yet, knock down of SNTA did neither affect proliferation nor viability of Hepa1-6 cells. The present study shows that SNTA protein levels are inversely related to TUBA8 protein expression in the hepatocyte cell line Hepa1-6.