转染ANGPTL-1基因对人皮肤成纤维细胞胶原表达的影响

目的探讨重组ANGPTL-1基因转染人皮肤成纤维细胞的可行性,以及转染转染后对细胞增殖速率和Ⅰ、Ⅲ型胶原合成的影响。方法体外重组ANGPTL-1基因,借助真核表达载体系统,转入体外培养的人皮肤成纤维细胞,流式细胞仪检测基因表达及细胞转染效率;RT-PCR比较转染前后ANGPTL-1 mRNA及Ⅰ、Ⅲ型胶原mRNA的表达。结果基因转染后,(63±7.1)%的被转染细胞表达目的基因;转染组ANGPTL-1 mRNA相对表达量较未转染组和转染空载体组明显增高(P<0.01,n=5);转染组Ⅰ、Ⅲ型胶原mRNA相对表达量较未转染组和转染空载体组均明显增高(P<0.01,n=5)。结论人皮肤成纤维细胞可作为ANGPTL-1转染的靶细胞,ANGPTL-1基因可能是促进Ⅰ、Ⅲ型胶原合成的重要基因之一。

染网茯皮的代用品

今年渔业生产将要更大的跃进，随着各种网具增多，染网所用的茯皮（栲皮）需用数量很大，但目前茯皮来源缺乏。各地发动群众找窍门，利用废物代替茯皮，如用茶壳，莲壳，马尾松，金钢刺，薯良等八种代用品，

Deletion of cagA gene of Helicobacter pylori by PCR products

AIM: Cytotoxin-associated protein (antigen) A (CagA) plays an important role in Helicobacter pylori(H pylori) pathogenesis.Our aim was to obtain cagA mutant strains by a new mutation method so as to better understand the mechanism of CagA in epithelial cells. METHODS: In contrast with the traditional method using suicide plasmid,we constructed cagA- mutant strains directly with PCR products. The constructed mutant clones grew on selective media and allelic exchange was confirmed by Southern blot. Furthermore, two different transformation methods, electroporation, and natural transformation, were compared with regard to the efficiency of recombination. RESULTS:The mutation by PCR products could be completed within 3-5 d, and the recombination rate by electroporation and natural transformation was 4.02×10-8 and 1.03×10-9 respectively. Mutation rate by electroporation (4.02×10-8) was far higher than by natural transformation (1.03×10-9) (P= 0.000<0.005). CONCLUSION: cagA- mutant strains have been constructed, which is important for further study on the function of CagA in epithelial cells.A mutation method by directly using PCR products has been proved successful with a much higher mutation rate, and is easier,especially when in combination with electroporation.This method could be widely used in gene deletion of H pylori.

The miRNAs of Herpes Simplex Virus(HSV)

Herpes simplex virus (HSV) is a group of common human pathogens with two serotypes HSV-1 and HSV-2.The prevalence of HSV is worldwide.It primarily infects humans through epithelial cells,when it introduces a latent infection into the nervous system.During viral latency,only a region known as the latency-associated transcript (LAT) is expressed.The discovery of HSV miRNAs helps to draw a larger picture of the infection and pathogenesis of the virus.This review summarizes miRNAs found in HSV-1 and HSV-2 so far.The functional studies of miRNAs in HSV to date indicate that they play a stage-specific role coordinated with viral proteins to maintain the virus life cycle.

Antifibrotic Effects of Genistein and Quercetin In Vitro

Objective: To study the antifibrotic effects of genistein(GE) and quercetin(QU) on rat hepatic stellate HSC-T6 cell proliferation stimulated with platelet-derived growth factor (PDGF), collagen synthesis and type I procollagen messenger RNA (mRNA) expression stimulated with transforming growth factor b1 (TGFb1). Methods: Cell proliferation was measured by crystal violet staining assay. Collagen synthesis was determined by 3H-proline incorporation assay. Type I procollagen mRNA level was determined by reverse transcription polymerase chain reaction (RT-PCR). Results: GE (25~70 mmolL-1) and QU (6.25~50 mmolL-1) concentration-dependently attenuated PDGF-driven HSC-T6 cell proliferative activity. TGFb1-stimulated collagen synthesis was also reduced. This was associated with a decrease in type I procollagen mRNA expression, indicating an effect at a pretranslational level. Conclusion: GE and QU may have therapeutic potential against liver fibrosis by regulating PDGF and TGFb1 actions.

STAPHYLOCOCCAL SCALDED SKIN SYNDROME: RETROSPECTIVE ANALYSIS OF 82 CASES

Objective To explore distinctive clinical manifestations and appropriate treatment, and assess prognosis of staphylococcal scalded skin syndrome （SSSS）. Methods A retrospective analysis was conducted of the data of 82 cases of SSSS hospitalized at Xinhua Hospital during the period from May 1993 to September 2003. Results The disease in all the 82 patients occurred in their first decade （mean 2.5 years）. Possible predisposing factors were found in 48 （58. 5% ）. Fever was present in 78 （95. 1% ）. Radial spokes of crusting around mouth were present in 80 （ 97. 6% ）. Erythema began on the face, especially around the mouth and eye in 63 （ 76. 8% ）. The course was acute in all cases and the eruptions quickly spread to the whole body within one day to two days. Of the 82 cases of SSSS, 47 were complete form of SSSS, 27 were abortive form of SSSS, and 8 were between the two forms. Staphylococcus aureus with positive staphylocoagulase was isolated from the possible primary infection sites including pharynx, eyelid, conjunctiva, nose, ear, and skin in 18 of 31 patients. Microbiological cultures of bullae and little pustulae developed after the onset were negative in 16 cases. All the 82 patients completely recovered after receiving antibiotic therapy （ ceftriaxone, oxacillin） alone or in combination with human immunoglobulin （IVIG） therapy. Additional IVIG therapy was used in those patients who had systemic involvements such as pneumonia, fever higher than 38. 5℃ or leukocytosis. Conclusion SSSS is a spectrum disease. Besides abortive and complete forms, presenting between the two forms a new form might be appeared in 8 cases who developed both scarlatiniform rash and flaccid bullae. The abortive form and complete form are usually misdiagnosed clinically. Radial spokes of crusting around mouth seem to be characteristic manifestation of SSSS. All the patients in this study had favorable prognosis after receiving prompt diagnosis and appropriate treatment.

THE FIRST CASE OF PROTOTHECOSIS ZOPFII IN CHINA

Objective. Report of first case of Protothecosis zopfii in China and causes the skin infection in the world. Method.By clinical and laboratory examinations to confirm the diagnosis and the response to treatment. By the review of literatures to confirm the first case of human skin infection in the world. Result.From the literatures and the clinical pictures, it is confirmed that this is the first case report of Protothecosis zopfii of skin in the world. Conclusion.The first case of Protothecosis zopfii in human being was reported and successfully treated with local infiltration of Diflucan (fluconazole) 3ml (2mg/ml)/week×4.

H pylori receptor MHC classⅡcontributes to the dynamic gastric epithelial apoptotic response

AIM： To investigate the role of MHC class Ⅱ in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS： After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class Ⅱ and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. RESULTS： Pretreatment of N87 cells with the anti-MHC class ⅡIgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class Ⅱ resulted in markedly reduced caspase activation, while simple ligation of MHC class Ⅱ did not. Crosslinking of MHC class Ⅱ also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-MHC class Ⅱ IgM blocked the recruitment of FADD to the cell surface. CONCLUSION： The ability of MHC class Ⅱ to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. The crosslinking of this molecule has anti-apoptotic effects during the earlier time points of Hpylori infection. This effect is possibly mediated by the ability of MHC class Ⅱ to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by Hpylori-infected gastric epithelial cells.

Adherence and invasion of mouse-adapted H pylori in different epithelial cell lines

AIM： To assess the adhesion and invasion abilities of different mouse adapted H py/or/strains in different cell lines in vitro and investigate their effects on the virulence factors cagA and vacA. METHODS： The adherence and invasion abilities of different N pylori strains in different epithelial cell lines were examined by the gentamycin protection assay. The null mutants of cagA and vacA were processed by direct PCR mutation method. The morphologic changes of different cell lines after N pylori attachment were examined by microscopy. RESULTS： The densities of adherence to and invasion into cells in vitro were different from those in the mouse infection experiments. 88-3887 strain could invade and adhere to cells stronger than SS1 and X47. All tested strains had better adhering and invasive abilities in SCG-7901 cell. CagA and vacA minus mutants had the same invasion and adherent abilities as their wild types. In all strains and cell lines tested, only AGS cell had the significant hummingbird phenotype after inoculation with the 88-3887 wild-type. CONCLUSION： Both the host cells and the bacteria play important parts in the invasion and adhesion abilities of Hpylori. CagA and VacA are not related to the ability of invasion and adhesion of Hpylori in different cell lines in vitro.

Elevation of vascular endothelial growth factor production and its effect on revascularization and function of graft islets in diabetic rats

AIM: To determine whether the elevated vascular endothelial growth factor (VEGF) expression produced by the transfected vascular endothelial cells (VECs) could stimulate angiogenesis of the graft islets and exert its effect on the graft function. METHODS: Thirty diabetic recipient rats were divided into three groups (n = 10 per group). In the control group,300 IEQ islets were transplanted in each rat under the capsule of the right kidney,which were considered as marginal grafts. In the VEC group,VEC together with the islets were transplanted in each rat. In the VEGF group,VEC transfected by pIRES2-EGFP/ VEGF165 plasmid and the islets were transplanted in each rat. Blood glucose and insulin levels were evaluated every other day after operation. Intravenous glucose tolerance test (IVGTT) was performed 10 d after the transplantation. Hematoxylin and eosin (HE) staining was used to evaluate the histological features of the graft islets. Immunohistochemical staining was used to detect insulin-6,VEGF and CD34 (MVD) expression in the graft islets. RESULTS: Blood glucose and insulin levels in the VEGF group restored to normal 3 d after transplantation. In contrast,diabetic rats receiving the same islets with or without normal VECs displayed moderate hyperglycemia and insulin,without a significant difference between these two groups. IVGTT showed that both the amplitude of blood glucose induction and the kinetics of blood glucose in the VEGF group restored to normal after transplantation. H&E and immunohistochemical staining showed the presence of a large amount of graft islets under the capsule of the kidney,which were positively stained with insulin-6 and VEGF antibodies in the VEGF group. In the cell masses,CD34-stained VECs were observed. The similar masses were also seen in the other two groups,but with a fewer positive cells stained with insulin-6 and CD34 antibodies. No VEGF-positive cells appeared in these groups. Microvessel density (MVD) was significantly higher in the VEGF group compared to the other two groups. CONCLUSION: Elevated VEGF production by trans-fected vascular endothelial cells in the site of islet transplantation stimulates angiogenesis of the islet grafts. The accelerated islet revascularization in early stage could improve the outcome of islet transplantation,and enhance the graft survival.

Reliable Analytic Strategy to Correlate the Morphological and Cytological Parameters on Lupinus termis L, against Fusarium oxysporum Infection

Many analytic strategies have emerged to estimate plant responses to Fusarium wilt. The demand for fast and reliable method （diagnosis, prediction） to determine isolate strength accurately is not established yet. Early determination of pathogen strength helps in plant medication. The aim of this study was to develop a faster strategy and method for early determination of fungal isolates strength in correlation to plant response. Till now, the scientists have no consensus on the most correlated parameters that could express wilt precisely. In this study, 30 isolates of Fusarium oxysporum isolated from Lupinus termis L. were used to provide an explicit image about the real strength of Fusarium isolates and its impact on the plant. Wilting percentage ranged from 26.67% to 93.33% of the infected plants depending on isolate virulence. Some of cellular, morphological and physical measurements were conducted on 8 out of 30 isolates, including root （length, fresh weight （FW） and dry weight （DW））, nodules （water content （WC）, FW, DW）, stem （height, WC, FW, DW）, total leaves/plant （WC, FW, DW） and the fourth leaf （WC, FW, DW, leaf area, epidermal cell area, epidermal cell number, succulence）. Hierarchical clustering was used to determine the variance between the isolates. Detrended correspondence analysis （DCA） and canonical correspondence analysis （CCA） were used to determine the most important growth parameters that could express wilting accurately. The CCA results showed that most of the measured parameters on the fourth leaf, except for leaf epidermal cell number, were highly and positively correlated to wilt. That makes these specific parameters valuable and sensitive for any changes in isolates strength. Accordingly, a mathematical model was created to be helpful in the quick determination of isolate strength and precise medication.

Clinical significance of HBME-1,Galectin-3,and CK19 expression and the status of BRAF mutation in papillary thyroid carcinoma

Objective The aim of this study was to explore the clinical significance of the expression of proteins human bone marrow endothelial cell markers(HBME-1), Galectin-3, and cytokeratin19(CK19), as well as the status of v-raf murine sarcoma viral oncogene homolog B1(BRAF) mutation in papillary thyroid carcinoma(PTC). Methods Immunohistochemical staining was performed in 82 specimens each of PTC and papillary benign lesions to detect the expression of HBME-1, Galectin-3, and CK19. Polymerase chain reaction(PCR) and gene sequencing were performed on 60 specimens each of PTC and papillary benign lesions to detect the status of BRAF mutation. Results The positive expression ratios of HBME-1, Galectin-3, and CK19 in PTC were 98.8%, 97.6% and 100% respectively, which were significantly higher than the expressions in papillary benign lesions(P < 0.05). No significant relationship was observed between the expression of these makers and the clinicopathological features of PTC. The sensitivity of co-expression of HBME-1 and CK19 or HBME-1 and Galectin-3 as diagnostic criteria of PTC was 99.9%, with a specificity of 95.4%. BRAF mutation was detected in 40 of 60 PTC(66.7%) specimens. There was a statistical difference in BRAF mutations between PTC and papillary benign lesions(P < 0.05); there were no associations between BRAF mutation and the clinicopathological features of PTC. Conclusion Combined immunohistochemical staining of HBME-1, Galectin-3, and CK19 can further improve the sensitivity and specificity of differential diagnosis of PTC. BRAF mutation is a significant genetic event, which may have diagnostic value for PTC.

Different morphologic features of rat cochlea progenitor spheres and their implications

Objective: To detect the different morphologic features, developmental regulation, potential of proliferation and differentiation of neonatal rat cochlea progenitor spheres. Methods: We isolated the cochlea sensory epithelium cells from neonatal rats and cultured them in nonadherent conditions to acquire different morphologic spheres. Then we observed the diameter and compositional change of cell colonies in distinct sphere types on day 3, 6, 9 and 12, and summarized the regularity of development and their conversion. We also detected the proliferative activity of distinct spheres by immunohistochemical staining of Abcg2, Nestin and BrdU. After induced spontaneous differentiation, the spheres were detected in the changes of the marker of hair cell, MyosinVIIA; by immunocytochemical staining, we revealed the potential of how different spheres were converted into hair cell-like cells. Results: The acquired three types of suspended spheres are solid, transitional, and hollow. There's morphologic significance among them and they can covert into the other type of spheres among them. The ability of self-renewing and proliferation in distinct spheres vary and all of them have the potential of spontaneously differentiation into hair cell-like cells. Conclusion: All the type of spheres not only has the potential of proliferation and differentiation, but also hasthe potential of spontaneous differentiation into hair cell-like cells. Distinct types of cell spheres neither originate from different progenitor cell subcolonies nor are different stages of the same cell spheres. Solid spheres are most practically useful.

Impact of Air Pollution on Skin

Recent articles reviewed the molecular damaging pathway of various pollutants especially PM2.5 particles and polycylic aromatic hydrocarbon. AhR （aryl hydrocarbon receptor） pathway, tobacco-similar pathway, oxidation, as well as epigenetics pathway were reviewed. Based on the working mechanism, the reported study method and methodology used in Ashland lab were reviewed, including cell level experiment, through reconstructed human skin to in vivo study.

EPIDERMAL GROWTH FACTOR PREVENTS INCREASED PERMEABILITY AND BACTERIAL TRANSLOCATION IN RATS WITH ACUTE PANCREATITIS

To evaluate the effects of epidermal growth factor (EGF) on intestinal permeability and bacterial translocation in rats with acute pancreatitis during total parenteral nutrition (TPN). Methods. Thirty- two male Sprague- Dawley rats that underwent injection of 3.5％ sodium taurocholate solution into the pancreatic duct were randomly divided into one of the following two groups: (1) received only TPN (control group) or (2) received TPN with EGF at a dose of 0.2 mg· kg- 1· day- 1 (Egf group). On fifth day of total parenteral nutrition, samples from mesenteric lymph nodes, pancreas, liver and spleen were harvested for cultures. Water, protein and DNA content in jejunal mucosa were determined. D- xylose and fluorescein isothiocyanate (FITC)- dextran were instilled into the lumen of a ligated segament of small intestine. Thirty minutes later, superior mesenteric vein D- xylose and plasma FITC- dextran concentration were measured. Results. Positive cultures in liver and spleen, as well as FITC- dextran concentration in the Egf group were significantly lower than in the control group. Protein and DNA content in jejunal mucosa in the Egf group were significantly higher than in the control group. Conclusion. The results indicate that EGF may prevent increased intestinal permeability and bacterial translocation in rats with acute pancreatitis during TPN.

Laboratory Limits on Dermatophyte Diagnosis in Rabbits with Clinical Lesions

Dermatophyte infection or ringworm is a superficial cutaneous infection with one or more of the fungal species of the keratinophilic genera Microsporum, Trichophyton, or Epidermophyton and is a zoonosis with a great impact on public health. Dermatophytes were identified from rabbit sample cultures submitted to mycological examination in the Laboratory of Microbiology of the University of Tr^-os-Montes e Alto Douro, Vila Real, Portugal. All samples were collected from suspected clinical cases. Dermatophytes were cultured from 4 of the 55 specimens （7.3%）. The dermatophytes isolated were Trichophyton mentagrophytes var. mentagrophytes （1.8%） and Microsporum gypseum （5.5%）. Microscopic examination was negative in all specimens. In this work, Scopulariopsis spp., a contaminant mould, was identified in 13 specimens （23.6%）. The proportion of positive samples in relation to the number of samples examined from cases suspected was very low. As all samples were collected from rabbits with compatible signs, we presume that the low prevalence of isolation was due to laboratory constraints on dermatophytes diagnosis.

Helicobacter pylori neutrophil activating protein as target for new drugs against H.pylori inflammation

Helicobacter pylori(H.pylori) infection is among the most common human infections and the major risk factor for peptic ulcer disease and gastric cancer.With-in this work we present the implication of C-terminal region of H.pylori neutrophil activating protein in the stimulation of neutrophil activation as well as the evi-dence that the C-terminal region of H.pylori activating protein is indispensable for neutrophil adhesion to endothelial cells,a step necessary to H.pylori inflammation.In addition we show that arabino galactan proteins derived from chios mastic gum,the natural resin of the plant Pistacia lentiscus var.Chia inhibit neutrophil activation in vitro.

Characterization of a New Methodology Based on the Intensity of Skin Staining of Ornamental Fish with Applications in Nutrition

The colors presented in ornamental fish determine their acceptability in the market, which is one of the main factors which determine their price. Selecting the fish by only looking at them makes the result analysis based in statistics difficult. Thus, it has become necessary to develop a method of quantification for practical use, which will apply minimal stress to the fish while being economically feasible. The proposed method uses a technique which converts the colors of interest to numerical indexes usable in statistical methods of higher precision. The results increase the abilities of fish nutrition researchers aiming to intensify the color characteristics of fish through selective breeding and genetic engineering with the intent of reliably achieving the desired visual characteristics for commercialization.

Growth-inhibitory effects of MOB2 on human hepatic carcinoma cell line SMMC-7721

AIM:To investigate the growth-inhibiting and apoptosis-inducing effects of the gene MOB2 on human hepatic carcinoma cell line SMMC-7721.METHODS:The full-length cDNA of the MOB2 gene was amplified from human umbilical vein endothelial cells.The correct full-length MOB2 cDNA was subcloned into the eukaryotic expression vector pEGFP-C1.After lipofection of the MOB2 gene into cancer cells,the levels of MOB2 protein in the cancer cells were detected by immunoblotting.To transfect the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells,the cells were cultured in Dulbecco's Modified Eagle'sMedium with 10% fetal calf serum and glutamine,and then mixed with liposomes,Lipofectamine 2000 and the plasmid vector pEGFP-CI-MOB2.RESULTS:We observed the growth and proliferation of SMMC-7721 cells containing pEGFP-CI-MOB2 and analyzed their apoptosis and growth cycle phases by flow cytometry.We successfully transfected the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells and screened for a single clone cell containing MOB2.After transfection,MOB2 enhanced growth suppression,induced apoptosis,increased the ratio of G0/G1,significantly inhibited the advance of cell cycle phase,and arrested cells in G0/G1 phase.CONCLUSION:MOB2 overexpression induces apoptosis and inhibits the growth of human hepatic cancer cells,which may be useful in gene therapy for hepatic carcinoma.

Adenosine:An immune modulator of inflammatory bowel diseases

Inflammatory bowel disease(IBD)is a common and lifelong disabling gastrointestinal disease.Emerging treatments are being developed to target inflammatory cytokines which initiate and perpetuate the immune response.Adenosine is an important modulator of inflammation and its anti-inflammatory effects have been well established in humans as well as in animal models.High extracellular adenosine suppresses and resolves chronic inflammation in IBD models.High extracellular adenosine levels could be achieved by enhanced adenosine absorption and increased de novo synthesis.Increased adenosine concentration leads to activation of the A2a receptor on the cell surface of immune and epithelial cells that would be a potential therapeutic target for chronic intestinal inflammation. Adenosine is transported via concentrative nucleoside transporter and equilibrative nucleoside transporter transporters that are localized in apical and basolateral membranes of intestinal epithelial cells,respectively. Increased extracellular adenosine levels activate the A2a receptor,which would reduce cytokines responsible for chronic inflammation.