血管紧张素Ⅱ在缺氧诱导的人肺成纤维细胞表型转化及胶原合成中的作用

目的:探讨血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)在缺氧诱导的人肺成纤维细胞(human lung fibroblast,HLF)表型转化及胶原合成中的作用。方法:在缺氧条件下培养HLF-1细胞株,将细胞分为AngⅡ组、AngⅡ+替米沙坦(TST)组和对照组。采用免疫荧光法检测HLF-1细胞α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达水平;采用Western blot法检测HLF-1细胞Ⅰ型胶原(collagen typeⅠ,Col-Ⅰ)蛋白的表达水平。结果:AngⅡ组HLF-1细胞α-SMA和Col-Ⅰ蛋白的表达水平较对照组明显上调,AngⅡ+TST组α-SMA和Col-Ⅰ蛋白的表达水平较AngⅡ组明显下降。结论:AngⅡ/血管紧张素Ⅱ1型受体信号通路可诱导缺氧性HLF表型转化以及胶原合成。

生长因子受体反义基因逆转肝癌细胞的恶性表型

目的应用反义技术原理将生长因子受体反义基因,导入人肝癌SMMC-7721细胞,阻断肝癌细胞生长因子与其受体的相互作用,研究其对肝癌细胞恶性表型的逆转作用。方法构建人胰岛素样生长因子Ⅱ型受体(IGF-ⅡR)正、反义基因重组真核表达质粒,用磷酸钙-DNA共沉淀方法转染人肝癌细胞株SMMC-7721,经G418培养基筛选得到稳定的抗性细胞。采用Northern杂交检测反义基因转染细胞内源性IGF-ⅡR基因mRNA转录的抑制,同时观察转染细胞克隆形成率,细胞生长曲线和软琼脂生长能力的变化。结果 IGF-ⅡR反义基因转染后,肝癌细胞的内源性IGF-ⅡR基因mRNA转录受到有效抑制;各组转染细胞克隆形成率分别为:空载体组22.0±3.6,正义组13.3±2.1,反义组4.3±2.1,反义基因转染细胞克隆形成率明显降低;软琼脂培养基细胞集落形成数为:SMMC-7721组254.8±15.9,空载体组196.4±30.4。正义组99.4±23.0,反义组0.0±0.0,说明反义基因转染细胞失去在软琼脂上生长的能力;但细胞生长曲线无明显变化。结论应用反义策略干扰肝癌细胞生长因子受体基因的异常高表达状态,可以有效阻断配体与受体间作用的生长刺激信号环路,一定程度地抑制肝癌细胞的恶性表型。

Cavitary Pulmonary Metastases: CT Features and Their Correlation with the Pathology of the Primary Malignancy

Objective: To study CT features of cavitary pulmonary metastases and to investigate the pos- sible relationship between CT features and the pathology of the primary lesions. Methods: CT ?ndings of 131 cavitary metastatic nodules in 40 patients with pathologically-proved pulmonary metastases were retrospectively analyzed. A comparison between CT signs and the pathologic types of the primary tumors was made. Results: Cavitary metastases and multiple solid nodules coexisted in all patients. Cavitary metastases presented as bubble (n=41), irregular (n=33), cystic (n=26) or small circular (n=31) cavities, with even (n=61) or uneven (n=70) thickness of the cavity wall. Of 131 cavitary nodules, diameter less than 15 mm was seen in 44, between 15–25 mm in 66, 25–40 mm in 17 and larger than 40 mm in 4 respectively. And the wall thickness of the cavity below 4 mm, between 4–15 mm and over 15 mm was respectively seen in 69, 44 and 18 metastatic nodules. Cavitary pulmonary metastases mainly occurred in patients whose primary malignancy was squamous cell carcinoma (n=13) or adenocarcinoma (n=22). Both squamous cell carcinoma and adenocarcinoma had its own CT characteristics. The occurrence of cavity bore no relationship to its site in the lung. Conclusion: Cavitary pulmonary metastases carries certain CT features and its occurrence is related to the pathologic type of the primary malignancy.

EGFR antisense RNA blocks expression of the epidermal growth factor receptor and partially reverse the malignant phenotype of human breast cancer MDA-MB-231 cells

The effects of human EGFR to the malignant phenotype of human breast cancer cell line MDA-MB-231 were investigated experimentally. A retroviral vector containing a 5'1350bp fragment of the human EGFR cDNA in the antisense orientation was transfected into targeted cells by lipofectamine. The effects on cell proliferation, cell cycle and adherent ability to extracellular matrix (ECM) components were studied after the expression of antisense transcripts to EGFR 5'1350bp fragment in target cells. In vitro studies showed that the growth ability of the transfected cells was partialy inhibited in comparison to parental cells and to cells transfected with the plasmid containing the neomycin resistance gene only. It was found that EGF (10ng/ml) had an augmenation effect on the growth of transfected MDA-AS10 cells but not MDA-MB-231 cells.Flow cytometric analysis showed that the cell cycle of the transfected cells was abnormal with a decrease of cells in G2/M and S phases and an increase of cells in G1 phase,indicating a blockage in phase G1. Immunofluorescence of EGFR expression in transfectants stained with an antiEGFR antibody was decreased and their growth in soft agarose was also severely impaired. The transfected cells showed less adherence to laminin (LN) and fibronectin (FN). In short, EGFR antisense RNA decreases the expression of EGFR on MDA-MB-231 cells and partially reverses their malignant phenotype as well.Effects of antisense EGFR on human breast cancer MDA-MB-231 cells

High miR-196a levels promote the oncogenic phenotype of colorectal cancer cells

AIM： To analyze the relevance of the microRNA miR-196a for colorectal oncogenesis.METHODS： The impact of miR-196a on the restriction targets HoxA7, HoxBS, HoxC8 and HoxD8 was analyzed by reverse transcription polymerase chain reaction（RT-PCR） after transient transfection of SW480 cancer cells. The miR-196a transcription profile in colorectalcancer samples, mucosa samples and diverse cancercell lines was quantified by RT-PCR. Transiently miR-196a-transfected colorectal cancer cells were used for diverse functional assays in vitro and for a xenograft lung metastasis model in vivo.RESULTS： HoxA7, HoxB8, HoxC8 and HoxD8 wererestricted by miR-196a in a dose-dependent andgene-specific manner. High levels of miR-196aactivated the AKT signaling pathway as indicated byincreased phosphorylation of AKT. In addition, highlevels of miR-196a promoted cancer cell detachment,migration, invasion and chemosensitivity towardsplatin derivatives but did not impact on proliferationor apoptosis. Furthermore, miR-196a increased thedevelopment of lung metastases in mice after tail veininjection.

Upreguiation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells

AIM： To study the changes of human telomerase reverse transcriptase （hTERT） mRNA expression in human hepatocarcinoma cell lines （HepG2） and cholangiocarcinoma cell lines （QBC939） after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis. METHODS： HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein （EGFP） coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency. Cells were harvested and total RNA was extracted using TRIzol reagent. The expression of hTERT mRNA in HepG2 and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting. RESULTS： Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939 cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector. Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939 cells only when transfected with HBx gene. CONCLUSION： HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection. 2005 The WJG Press and Elsevier Inc. All rights reserved

First-principles Study of Single Tin-phthalocyanine Molecule on Ag（111） Surface

Adsorption behavior and electronic structure of tin-phthalocyanine (SnPc) on Ag(111) surface with Sn-up and Sn-down conformations are investigated using first-principles calculations. Two predicted adsorption configurations agree well with the experimentally determined structures. SnPc molecule energetically prefers to adsorb on Ag(111) surface with Sn-down conformation. The energy required to move the central Sn atom through the frame of a phthalocyanine molecule, switching from the Sn-up to Sn-down conformation, is about 1.68 eV. The simulated scanning tunneling microscopy images reproduce the main features of experimental observations. Moreover, the experimentally proposed hole attachment mechanism is verified based on the calculated density of states of SnPc on Ag(111) with three different adsorption configurations.

Are polymorphisms of N-acetyltransferase genes susceptible to primary liver cancer in Luoyang, China?

AIM: To identify whether the polymorphisms of the Nacetyltransferase (NAT) genes are susceptible to primary liver cancer (PLC) in Luoyang, a PLC low-incidence area of China.METHODS: The NAT1 and NAT2 genotypes of 96 PLC cases and 173 controls were determined by PCR-RFLP.Both interaction between NAT1 or NAT2 and environmental risk factors were analyzed based on case control study.RESULTS: Compared to the control group, the frequencies of alleles NAT1*3, NAT1*4, NAT1*10, NAT1*14B and alleles NAT2*4, NAT2*6, NAT2*7 in PLC group showed no statistically significant difference (x2 = 2.61 and 4.16,respectively, both P＞0.05). The frequencies of NAT1 genotypes NAT1*3/*3, NAT1*3/*4, NAT1*3/*10,NAT1*3/*14B, NAT1*4/*4, NAT1*4/*10, NAT1*4/*14B,NAT1*10/*10, NAT1*10/*14B, and NAT2 genotypes NAT2*4/*4, NAT2*4/*6, NAT2*4/*7, NAT2*6/*6,NAT2*6/*7 and NAT2*7/*7 also had no statistically significant difference between the two groups (x2 = 11.86 and 2.94respectively both, P＞0.05). Neither the frequencies of rapid and slow NAT1 acetylators nor the frequencies of rapid and slow NAT2 acetylators were significantly different between the two groups (x2 = 0.598 and 0.44,respectively, both P＞0.05). The interaction betweenNAT1*10 and occupational exposures was found significant with an odds ratio of 3.40 (x2 = 8.42, P = 0.004,OR 95%CI:1.03-11.22). But no interaction was found between NAT2 and any environmental risk factors.CONCLUSION: The polymorphisms of NAT1 and NAT2are not susceptible to PLC in Luoyang. Allele NAT1*10interacts with occupational exposures.

Microhabitat Effect on Iron Distribution and Transfer in Carex pseudocuraica in Sanjiang Plain Wetlands

Ten clonal units of Carex pseudocuraica growing in four different microhabitats (perennial flooded ditch water,perennial flooded ditch sediment,seasonal flooded ditch sediment and perennial flooded soil) of the Sanjiang Plain,Northeast China,were collected randomly for phenotypic plasticity analysis.Iron content,chemical and physical properties of substrates and the total Fe of nine plant modules were measured as well.The results show that the performance of the C.pseudocuraica is affected by the microhabitat,with the greatest performance score in perennial flooded ditch water,and the lowest in perennial flooded soil.The biomass allocation indexes indicate that much more mass is allocated to stems and roots to expand colonization area.The distribution of the total Fe in plant modules appears as pyramids from the tip to the root,while marked differences are observed in the distribution proportion of stems,tillering nodes and roots that are allometrically growing.Iron transfer from substrates to the plant is mainly controlled by the substrate type.The differences of iron distribution and transfer in the plant in different microhabitats are attributed to the iron contents of the substrates as well as the phenotypic plasticity of the plant.

Expression of Activated Epidermal Growth Factor Receptor and Transcription Factor E2F in Condyloma Accuminata

Objective: To study the expression of activated epi-dermal growth factor receptor (EGFR) and transcrip-tion factor E2F (E2F) in Condyloma Accuminata(CA)patients. Methods: Immunofluorescent techniques were usedto investigate the expression of activated EGFR andE2F in CA patients. Results: The expression of activated EGFR on themembrane of epithelial cells in CA lesions was sig-nificantly greater compared to expression levers inthe control group (P<0.01). Moreover, the co-expres-sion of activated EGFR and E2F was significantly in-creased compared to the control group (P<0.01). Conclusion: Our observations suggest that the in-crease in activated EGFR expression may stimulatehyperplasia in CA patients through the activation oftranscription factor E2F.

Macrophage secretory products induce an inflammatory phenotype in hepatocytes

AIM:To investigate the influence of macrophages on hepatocyte phenotype and function.METHODS:Macrophages were differentiated from THP-1 monocytes via phorbol myristate acetate stimulation and the effects of monocyte or macrophageconditioned medium on HepG2 mRNA and protein expression determined.The in vivo relevance of these findings was confirmed using liver biopsies from 147 patients with hepatitis C virus(HCV)infection.RESULTS:Conditioned media from macrophages,but not monocytes,induced a transient morphological change in hepatocytes associated with upregulation of vimentin(7.8±2.5-fold,P=0.045)and transforming growth factor(TGF)-β1(2.6±0.2-fold,P<0.001)and downregulation of epithelial cadherin(1.7±0.02-fold,P=0.017)mRNA expression.Microarray analysis revealed significant upregulation of lipocalin-2(17-fold,P <0.001)and pathways associated with inflammation,and substantial downregulation of pathways related to hepatocyte function.In patients with chronic HCV,realtime polymerase chain reaction and immunohistochemistry confirmed an increase in lipocalin-2 mRNA(F0 1.0 ±0.3,F1 2.2±0.2,F2 3.0±9.3,F3/4 4.0±0.8,P= 0.003)and protein expression(F1 1.0±0.5,F2 1.3± 0.4,F3/4 3.6±0.4,P=0.014)with increasing liver injury.High performance liquid chromatography-tandem mass spectrometry analysis identified elevated levels of matrix metalloproteinase(MMP)-9 in macrophageconditioned medium,and a chemical inhibitor of MMP-9 attenuated the change in morphology and mRNA expression of TGF-β1(2.9±0.2 vs 1.04±0.1,P<0.001) in macrophage-conditioned media treated HepG2 cells.In patients with chronic HCV infection,hepatic mRNA expression of CD163(F0 1.0±0.2,F1/2 2.8±0.3,F3/4 5.3±1.0,P=0.001)and MMP-9(F0 1.0±0.4,F1/2 2.8±0.3,F3/4 4.1±0.8,P=0.011)was significantly associated with increasing stage of fibrosis.CONCLUSION:Secreted macrophage products alter the phenotype and function of hepatocytes,with increased expression of inflammatory mediators,suggesting that hepatocytes actively participate in liver injury.

Role of H3K27 methylation in the regulation of IncRNA expression

Once thought to be transcriptional noise, large non-coding RNAs （IncRNAs） have recently been demonstrated to be functional molecules. The cell-type-specific expression patterns of lncRNAs suggest that their transcription may be regulated epigenetically. Using a custom-designed microarray, here we examine the expression profile of IncRNAs in embryonic stem （ES） cells, lineage-restricted neuronal progenitor cells, and terminally differentiated fibroblasts. In addition, we also analyze the relationship between their expression and their promoter H3K4 and H3K27 methyla- tion patterns. We find that numerous lncRNAs in these cell types undergo changes in the levels of expression and promoter H3K4me3 and H3K27me3. Interestingly, lncRNAs that are expressed at lower levels in ES cells exhibit higher levels of H3K27me3 at their promoters. Consistent with this result, knockdown of the H3K27me3 methyltransferase Ezh2 results in derepression of these IncRNAs in ES cells. Thus, our results establish a role for Ezh2-mediated H3K27 methylation in lncRNA silencing in ES cells and reveal that lncRNAs are subject to epigenetic regulation in a similar manner to that of the protein-coding genes.

The phenotype and activation status of regulatory T cells during Friend retrovirus infection

The suppressive capacity of regulatory T cells （Tregs） has been extensively studied and is well established for many diseases.The expansion,accumulation,and activation of Tregs in viral infections are of major interest in order to find ways to alter Treg functions for therapeutic benefit.Tregs are able to dampen effector T cell responses to viral infections and thereby contribute to the establishment of a chronic infection.In the Friend retrovirus （FV） mouse model,Tregs are known to expand in all infected organs.To better understand the characteristics of these Treg populations,their phenotype was analyzed in detail.During acute FV-infection,Tregs became activated in the spleen and bone marrow,as indicated by various T cell activation markers,such as CD43 and CD103.Interestingly,Tregs in the bone marrow,which contains the highest viral loads during acute infection,displayed greater levels of activation than Tregs from the spleen.Treg expansion was driven by proliferation but no FV-specific Tregs could be detected.Activated Tregs in FV-infection did not produce Granzyme B （GzmB） or tumor necrosis factor α （TNFα）,which are thought to be a potential mechanism for their suppressive activity.Furthermore,Tregs expressed inhibitory markers,such as TIM3,PD-1 and PD-L1.Blocking TIM3 and PD-L1 with antibodies during chronic FV-infection increased the numbers of activated Tregs.These data may have important implications for the understanding of Treg functions during chronic viral infections.

Multiple Ionic-Covalent Couplings in Molecules and Clusters

The electronic states of molecules made of electropositive and electronegative components result from the interference between the covalent configurations and the ionic configurations. This work shows complex aspects of these ionic-covalent couplings in small molecules such as Li2H, Li2F, and Li4F. The extension of this type of analysis to the adsorption of the electrophilic molecules on the metal clusters or on the metal surfaces is supposed to lead to a radically new interpretation of the observed physical and chemical properties.

Cell Type Specific Expression of CD80 （B7-1） Gene in Murine

The CD80 （B7-1） costimulatory molecule is expressed on the surface of B cells and its expression is transcriptionally upregulated upon stimuli. To identify the region of murine CD80 promoter that direct cell type specific gene expression, four promoters construct of CD80 gene were generated with DNA fragments fused to the GFP reporter gene. In the present study, significant promoter activity was detected with all four promoter constructs only in the murine B lymphocyte. Further, the CD80 promoter region extending from -3,005 bp to ＋273 bp in relation to the previously reported transcription start site, was identified as tissue specific region. Interestingly, the shortest 700 bp （-427/＋273） of promoter fragment was sufficient to direct the CD80 gene expression. The level of CD80 expression was also found to be modulated by exogenous stimuli in B lymphocyte. Additionally, it was demonstrated that the CD80 gene expression is regulated at the level of transcription where the inducible CD80 gene transcript was detected in B lymphocyte with increasing time.

Gene expression analysis of pancreatic cystic neoplasm in SV40Tag transgenic mice model

AIM： To study the gene expression changes in pancreatic cystic neoplasm in SV40Tag transgenic mice model and to provide information about the prevention, clinical diagnosis and therapy of pancreatic cancer. METHODS： Using the pBC-SV40Tag transgenic mice model of pancreatic cystic neoplasm, we studied the gene expression changes by applying high-density microarrays. Validation of part gene expression profiling data was performed using real-time PCR.RESULTS： By using high-density oligonucleotide microarray, of 14113 genes, 453 were increased and 760 decreased in pancreatic cystic neoplasm, including oncogenes, cell-cycle-related genes, signal transduction-related genes, skeleton-related genes and metabolism-related genes. Among these, we confirmed the changes in Igf, Shh and Wnt signal pathways with real-time PCR. The results of real-time PCR showed similar expression changes in gene chip.CONCLUSION： all the altered expression genes are associated with cell cycle, DNA damage and repair, signal pathway, and metabolism. SV40Tag may cooperate with several proteins in promoting tumorigenesis.

Effect of retinoid X receptor alpha (RXRα) transfection on the proliferation and phenotype of rat hepatic stellate cells in vitro

Objective To study the effect of retinoid X receptor alpha (RXRα) transfection plus treatment with the RXRα ligand, 9-cis-RA, on the proliferation and phenotype of platelet-derived growth factor (PDGF)-activated hepatic stellate cells (HSCs). Methods PDGF activated rat hepatic stellate cells were transfected with eukaryotic expression vector pcDNA3.1- human RXRα, and confirmed by Western blot. Proliferation of transfected HSC was assayed by bromodeoxyuridine (BrdU) incorporation as well as MTT, and the phenotype (α-smooth muscle actin, desmin) was observed by immunocytochemistry with image analysis. Results Transfection of the RXRα gene and treatment with ligand 9-cis-RA of PDGF-activated HSCs extended the increased expression of RXRα protein for at least 168 hours. Cell proliferation and expressions of alpha-smooth muscle actin (α-SMA) and desmin were blocked, compared with groups of sham-transfected, PDGF-activated, no transfection, no ligand treatment, and irrelevant ligand treated HSCs. Conclusion Transfection with the RXRα gene followed by 9-cis-RA ligand treatment will inhibit the proliferation and reverse the phenotype of activated HSC.

The effect of transposable elements on phenotypic variation： insights from plants to humans

Transposable elements(TEs), originally discovered in maize as controlling elements, are the main components of most eukaryotic genomes. TEs have been regarded as deleterious genomic parasites due to their ability to undergo massive amplification. However, TEs can regulate gene expression and alter phenotypes. Also, emerging findings demonstrate that TEs can establish and rewire gene regulatory networks by genetic and epigenetic mechanisms. In this review, we summarize the key roles of TEs in fine-tuning the regulation of gene expression leading to phenotypic plasticity in plants and humans, and the implications for adaption and natural selection.

Differentiation of rat adipose tissue-derived mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro

Objective： To differentiate rat adipose tissue-derived mesenchymal stem cells （ADSCs） into cells with a nucleus pulposus-like phenotype in vitro, so as to lay a foundation for the cell-based transplantation therapy of degenerated intervertebral discs. Methods： Rat ADSCs were isolated only from the subcutaneous inguinal region and purified by limited dilution. ADSCs of the third passages were analyzed by fluorescence activated cell sorter （FACS） to detect the cell surface markers （Sca-1, CD44, CD45, CDI lb）. To induce ADSCs to- wards a nucleus pulposus-like phenotype, ADSCs were immobilized in 3-dimensional alginate hydrogels and cultured in an inducing medium containing transforming growth factor-beta1 （TGF- β1） under hypoxia （2% O2）, while control groups under normoxia （21% O2） in alginate beads in medium with or without the presence of TGF-β 1. Semiquantitative reverse transcription polymerase chain reaction （RT-PCR） was carried out to evaluate phenotypic and biosynthetic activities in the process of differentiation. Meanwhile, Alcian blue staining were used to detect the formation of sulfated glycosaminoglycans （GAGs） in the differentiated cells. Results： The purified ADSCs were fibroblast-like and proliferated rapidly in vitro. The flow cytometry showed that ADSCs were positive for Sca-1 and CD44, negative for CD45 and CD11b. The results of RT-PCR manifested that the gene expressions of Sox-9, aggrecan and collagen Ⅱ, which were chondrocyte specific, were upregulated in medium containing TGF-β1 under hypoxia （2% O2）. Likewise, gene expression of HIF-1 a, which was characteristics of in- tervertebral discs, was also upregulated. Simultaneously, Alcian blue staining exhibited the formation of many GAGs. Conclusions： The approach in our experiment is a simple and effective way to acquire a large quantity of homogenous ADSCs. Rat ADSCs can be differentiated into nucleus pulposus-like cells. ADSCs may replace bone marrow mesenchymal stem cells as a new kind of seed cells in regeneration of degenerated intervertebral discs using cell transolantation therarw.