人体肝癌细胞急性低氧及低氧习服差异表达基因分析

本文分析了人体肝癌细胞(HepG2)急性低氧处理以及低氧习服处理后基因表达谱的改变。急性低氧处理为细胞在1％氧气中培养48h,低氧习服处理为细胞在1％氧气中培养24h,常氧培养24h,以此作为一个周期,重复6个周期。联合应用抑制消减杂交技术和cDNA芯片技术,筛选HepG2细胞经急性低氧处理与正常培养细胞相比差异表达的基因,以及经低氧习服处理细胞与正常培养细胞相比差异表达的基因。结果显示,HepG2细胞经急性低氧处理与在常氧条件下培养相比,差异表达的基因有37个,表达水平全部表现为下调,其中包括参与细胞周期、细胞应激、细胞信号转导、细胞骨架形成、转录相关蛋白及细胞代谢相关蛋白的基因,1个未知基因序列、4个EST序列、5个线粒体蛋白基因,另外有功能不明的蛋白质基因12个。低氧习服处理的细胞与常氧条件下培养的细胞相比,差异表达的基因有6个,其中包括两个线粒体蛋白基因、金属蛋白酶1基因、转铁蛋白基因、Thymosin ．beta-4和TPT1基因。其中线粒体蛋白ND4、转铁蛋白、Thymosin.beta-4和TPT1基因的表达呈上调,线粒体NDl及金属蛋白酶1基因的表达水平呈下调。经低氧习服处理后,细胞低氧耐受力提高,低氧习服处理细胞基因的表达与急性低氧处理细胞和正常培养细胞的基因表达不同,这种变化可能与低氧习服细胞低氧耐受力的增强有关。     

Induction of phospholipase A2 gene expression in human hepatoma cells by mediators of the acute phase response.

Serum levels of phospholipase A2 (PLA2) activity have been shown to be elevated in cases of septic shock and rheumatoid arthritis. The cellular origin of serum PLA2, however, is not known. In this report, we demonstrate that human group II PLA2 expression and secretion are induced in hepatoma cells (HepG2) following treatment with interleukin-6 (IL-6), tumor necrosis factor (TNF), and interleukin-1 (IL-1). Of the three cytokines, IL-6 is the most potent. Significant synergy is observed between IL-6 and IL-1 and between IL-6 and TNF, but not between IL-1 and TNF. PLA2 induction does not occur in human YT cells, which are known to have receptors for both IL-1 and IL-6, indicating that the regulatory mechanism involved is cell type-specific. The results of RNA blot analysis indicate that the PLA2 gene is regulated in HepG2 cells at the pretranslational level. Induction of PLA2 synthesis in HepG2 cells in response to these cytokines resembles the induction of the acute phase plasma proteins which are synthesized in cultured hepatocytes and hepatoma cells following exposure to the same cytokines and in liver in response to inflammation and infection. In addition, a putative IL-6-responsive element, which is homologous to a similar element found in several acute phase genes, is present in the 5'-promoter-proximal region of the PLA2 gene. These results suggest that serum PLA2 is synthesized in and secreted from liver cells in response to inflammatory stimuli, mediated primarily by IL-6, and therefore should be classified as an acute phase protein.     

Identification of major cellular proteins synthesized in response to interleukin-1 and interleukin-6 in human hepatoma HepG2 cells

Interleukin-1 (IL-1) and interleukin-6 (IL-6) are principal proinflammatory cytokines inducing the acute phase response of various tissues, including liver. Cultured human hepatoma HepG2 cells were stimulated with IL-1 (10 ng/ml) and IL-6 (10 ng/ml). After 24 h the cells were collected and disrupted by sonication in a lysis buffer containing 8M urea. The extracted cellular proteins were separated by 2D polyacrylamide gel electrophoresis. The gels were stained with Coomassie Brilliant Blue R-250 and the protein spots showing different intensities in comparison to control (unstimulated) cells were excised and subjected to analysis by LC-MS/MS. Alternatively, proteins were stained with SYPRO Ruby. These differentially expressed proteins include seven up-regulated and two down-regulated intracellular proteins of various functions. The identification of three cytokine-responsive proteins was confirmed by biosynthetic labeling with [35S]methionine after incubation of HepG2 cells, and by western blot with specific antisera.     

Multiplex Genotyping of Cytokine Gene SNPs Using Fluorescence Bead Array

Single nucleotide polymorphisms (SNPs) of genes that affect cytokine production and function are known to influence the susceptibility and progression of immune-related conditions such as infection, autoimmune diseases, transplantation, and cancer. We established a multiplex genotyping method to analyze the SNPs of cytokine genes by combining the multiplex PCR and bead array platform. Thirteen cytokine gene regions, including 20 SNPs, were amplified, and allele-specific primer extension was performed in a single tube. High-quality allele-specific primers were selected for signals greater than 1000 median fluorescence intensity (MFI) for positive alleles, and less than 500 MFI for negative alleles. To select and improve the extension primers, modifications for the reverse direction, length or refractory were performed. 24 primers in the forward or reverse direction step and 12 primers in length or refractory modifications were selected and showed high concordance with results by nucleotide sequencing. Among the 13 candidate cytokine genes, the SNPs of 12 cytokine genes, including IL-1α, IL-1R, IL-1RA, IL-1β, IL-2, IL-4, IL-4Rα, IL-6, IL-10, IL-12, TGF-β1, and TNF-α, were successfully defined with the selected allele-specific primers in healthy Korean subjects. Our genotyping system provides a fast and accurate detection for SNPs of multiple cytokine genes to investigate their association with immune-related diseases and transplantation outcomes.     

Human hepatocyte-stimulating factor-III and interleukin-6 are structurally and immunologically distinct but regulate the production of the same acute phase plasma proteins

Human squamous carcinoma (COLO-16) cells synthesize and secrete hepatocyte-stimulating factor-III (HSF-III), a glycoprotein with Mr = 39,000, which stimulates the synthesis of several acute phase plasma proteins in human hepatoma (HepG2) cells. The qualitative response of HepG2 cells to HSF-III is essentially the same as that elicited by human recombinant interleukin-6 (IL-6). Although similar in hepatocyte-stimulating activity, HSF-III and IL-6 are distinct molecules which differ not only in size and charge but also in immunologic properties: no cross-recognition of HSF-III and IL-6 occurs using neutralizing antibodies against IL-6 and HSF-III, respectively. In addition, Northern blot hybridization of IL-6 cDNA to mRNA from COLO-16 cells revealed no detectable IL-6 message. HSF-III does not compete for binding to the IL-6 receptors suggesting that HepG2 cells carry receptors specific for each hormone. Both receptor types may trigger similar intracellular processes explaining the identical regulation of acute phase protein expression.     

Induction of Ym1/2 in mouse bone marrow-derived mast cells by IL-4 and identification of Ym1/2 in connective tissue type-like mast cells derived from bone marrow cells cultured with IL-4 and stem cell factor

Mast cells play an important role in allergic inflammation by releasing various bioactive mediators. The function of mast cells is enhanced by various stimuli, partly due to the induction of specific genes and their products. Although many inducible genes have been identified, a significant number of genes remain to be identified. Therefore, this study used PCR-selected cDNA subtraction to establish the profile of induced genes in the connective tissue (CT) type-like mast cells derived from bone marrow cells cultured in the presence of IL-4 and stem cell factor. Two hundred and fifty cDNA clones were obtained from the CT type-like mast cells by PCR-selected cDNA subtraction. Among them, Ym1/2, a chitinase-like protein, is one of the most abundantly induced genes. Ym1 is produced by activated macrophages in a parasitic infection, whereas its isotype, Ym2, is highly upregulated in allergic lung disease. In order to differentiate which isotype is expressed in bone marrow cells, specific primers for bone marrow-derived mast cells (BMMC), and CT type-like mast cells were used for RT-PCR. The results showed that Ym1 was constitutively expressed in bone marrow cells and gradually decreased in the presence of IL-3, whereas Ym2 was induced only in the presence of IL-4. CT type-like mast cells from bone marrow cells expressed Ym1 throughout the culture period and Ym2 was induced only by the addition of IL-4 into BMMC, indicating that IL-4 is essential for the expression of Ym1/2 genes.     

Soluble human interleukin-6-receptor modulates interleukin-6-dependent N-glycosylation of alpha 1-protease inhibitor secreted by HepG2 cells.

Interleukin-6 (IL-6) induces changes in gene expression and the N-glycosylation pattern of acute-phase proteins in hepatocytes. IL-6 exerts its action via a cell surface receptor complex consisting of an 80 kDa IL-6 binding protein (gp80) and a 130 kDa glycoprotein (gp130) involved in signal transduction. A genetically engineered gp80-derived soluble human IL-6-receptor (shIL-6-R) significantly enhanced the IL-6 effect on N-glycosylation changes (revealed by reactivity with the lectin-concanavalin A) of a1-protease inhibitor (PI) secreted by human hepatoma cells (HepG2). Stable transfection of IL-6-cDNA into HepG2 cells (HepG2-IL-6) resulting in constitutive secretion of 2 micrograms of IL-6 per 10(6) cells in 24 h led to a down-regulation of surface-bound gp80 and subsequent homologous desensitization of HepG2-IL-6 cells towards IL-6. Soluble human IL-6-R functionally substituted membrane-bound gp80 resulting in a reconstitution of responsiveness of HepG2-IL-6 cells.     

Interaction among hepatocyte-stimulating factors, interleukin 1, and glucocorticoids for regulation of acute phase plasma proteins in human hepatoma (HepG2) cells

Human hepatoma (HepG2) cells respond to unfractionated conditioned media of human squamous carcinoma (COLO-16) cells and lipopolysaccharide-stimulated human peripheral blood monocytes by increasing the synthesis of alpha 1-acid glycoprotein, haptoglobin, complement C3, alpha 1-antichymotrypsin, alpha 1-antitrypsin, and fibrinogen, while decreasing the synthesis of albumin. The regulation of the acute phase proteins is mediated by hepatocyte-stimulating factors (HSF) and interleukin 1 (IL-1) present in the conditioned medium. Purified HSF-I from COLO-16 cells stimulates preferentially alpha 1-acid glycoprotein synthesis, whereas COLO-HSF-II stimulates preferentially the synthesis of haptoglobin, fibrinogen, and alpha 1-antitrypsin. HSF from monocytes, which has been identified as interferon-beta 2 (B cell stimulating factor-2), displayed the same activity as COLO-HSF-II. Dexamethasone alone had no effect on acute phase plasma protein synthesis but enhanced the response to various HSF severalfold. IL-1 had a relatively low stimulatory activity on the synthesis of alpha 1-acid glycoprotein, haptoglobin, and alpha 1-antichymotrypsin but strongly reduced the basal expression of fibrinogen. The only synergistic action between IL-1 and HSF (or interferon-beta 2) was noted for the synthesis of alpha 1-acid glycoprotein. Tumor necrosis factor active on other hepatic cells failed to modulate significantly the expression of any plasma proteins in HepG2 cells. These studies showed that for an optimal HepG2-cell response a combination of HSF (or interferon-beta 2), IL-1, and dexamethasone is needed. This finding might indicate the identity of some of those hormones involved in regulation of the hepatic acute phase response in vivo.