Adolescent ; Adult ; Aged ; Chromogranin A ; metabolism ; Female ; Gastrointestinal Neoplasms ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Ki-67 Antigen ; metabolism ; Liver Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; Neuroendocrine Tumors ; metabolism ; pathology ; Pancreatic Neoplasms ; metabolism ; pathology ; Synaptophysin ; metabolism ; Young Adult

AIM: The study was undertaken to investigate the effort of Dexamethasone (DEX) on cultured rabbit corneal epithelial (RCE) cells and rabbit corneal epithelial wound healing.METHODS: For the in vitro experiments, primary cultures of RCE cells were used. DEX in different concentrations was added to cultured RCE cells. The effects were measured with tetrazolium salt (MTT)method and flow cytometry. For the in vivo wound-healing experiments, a central corneal deepithelialization was created and were treated with 0.1g/L DEX eyedrop randomly explain how randomly. Epithelial wound healing was evaluated clinically and analyzed histopathologically using light microscopy along with immunohistochemical staning and electronic microscopy.RESULTS: Less than 0.1g/L DEX didn't influence survival rate in cell culture conditions by MTT assay. Flow cytometric studies revealed that 0.1g/L DFX had no effect on cellular growth phase in cultured rabbit corneal epithelial cells. The mean time of the epithelial healing was significantly shorter in the DEX-treated group than in the control group at 24h. There were strong proliferative-cell-nuclear-antigen(PCNA) expressions in newly generated epithelial cells of both groups. The Dex-treated group had a more regular architecture of stromal lamella and significantly less inflammatory response than the control group under electronic microscopy.CONCLUSION: Less than 0.1g/L DEX had no inhibiting effect on cultured rabbit corneal epithelial cell growth.0.1g/L DEX eye drops can effectively promote epithelial growth and reduce inflammatory response, which may have useful clinical application at the early stage of corneal wound healing process.

OBJECTIVETo investigate the effects and regulatory mechanism of ILK on angiogenesis in hypertrophic scar.

METHODSThe human scar microvascular endothelial cells (HSMECs) were isolated from 6 patients' hypertrophic scar in vitro. The HSMECs with good condition in 2nd to 4th generation were selected as experimental objectives. (1) HSMECs were divided into the blank control group (treated with routine culture), negative control group (treated with only Lipofectamine 2000), LY294002 group (incubated with 50 nmol/L LY294002), ILK siRNA group (incubated with 20 nmol/L ILK siRNA). RT-PCR and Western Blot were used to detect the expression of ILK mRNA and its protein after transfecion for 48 h. (2) The digested HSMECs of four groups were resuspended with DMEM without serum and then seeded onto the upper compartment of transwell insert which contained complete medium in its lower compartment. The cell migration experiment was stopped in 10 h and then the migrated cells were counted to analyze the effects of different interventions on the migration ability of HSMECs. (3) The thawed ECMatrix was put into each well of pre-colled 48-well tissue culture plate, and then the plate was put into the incubator at 37 degrees C to make it to become gel. The HSMECs of four groups were seeded onto the surface of the ECMatrix gel and were put into incubator. Eight random view-fields per well should be valued by the sheet of pattern recognition about angiogenesis after 8 hours to evaluate the ability of angiogenesis in vitro between four groups.

RESULTS(1) The expression of ILK mRNA (ILK mRNA = 0.829 +/- 0.109, t = 13.151, P = 0.006) and protein (ILK protein = 0.096 +/- 0.049, t = 36.656, P = 0.000) were both inhibited obviously in ILK siRNA group compared with the blank control group (ILK mRNA = 0.829 +/- 0.109, ILK protein = 1). And, the expression of ILK in LY294002 group was slightly lower than that of black control group, but there was no statistical difference. (2) The number of migrated cells in ILK siRNA group (88.111 +/- 3.079) and LY294002 group (138. 667 +/- 2.404) were respectively lower than that in blank control group (322.333 +/- 3.712, P < 0. 05) in 10th hour. (3) Compared to blank control group (4.333 +/- 0.191), the ability of angiogenesis in vitro decreased significantly ILK siRNA group (2.625 +/- 0.125) and LY294002 group (3.125 +/- 0.250), in which, the vascular network structures were not formed perfectly in 8th hour (P < 0.05).

CONCLUSIONSThe ability of HSMECs' migration and angiogenesis in vitro are inhibited significantly when the expression of ILK is down-regulated. It reveals that ILK may play an role in the regulation of scar angiogenesis.

Cell Movement ; Cell Proliferation ; Chromones ; pharmacology ; Cicatrix, Hypertrophic ; enzymology ; pathology ; Endothelial Cells ; cytology ; drug effects ; Humans ; Lipids ; pharmacology ; Morpholines ; pharmacology ; Neovascularization, Pathologic ; etiology ; pathology ; Protein-Serine-Threonine Kinases ; genetics ; physiology ; RNA, Messenger ; analysis ; RNA, Small Interfering ; metabolism

Animals ; Cell Transformation, Neoplastic ; Cloning, Molecular ; Hepacivirus ; genetics ; immunology ; pathogenicity ; Hepatitis C Antigens ; toxicity ; Liver Neoplasms, Experimental ; pathology ; virology ; Male ; Oncogenic Viruses ; pathogenicity ; RNA, Messenger ; toxicity ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; toxicity ; Viral Core Proteins ; toxicity ; Virion ; pathogenicity

AIMIn order to discover new inhibitors and enhancers of nitric oxide synthase (NOS), an in vitro assay to determine NOS activity was established for high throughput screening.

METHODSThe activity of NOS was detected based on the change of nicotinamide-adenine dinucleotide phosphate (NADPH) concentration in the reaction system by the fluorescence density. The enzyme was prepared from bovine brain by gradient centrifugation. The reaction performed in black 96 well micro-plate with a final volume of 90 microL. Every factor which would affect the results such as the concentration of NADPH, L-arginine (L-Arg, used as substrate) and enzyme protein was optimized in different conditions. At last, 5,600 samples (compounds and extracts) were screened by the method.

RESULTSThe test signal (fluorescence density) in the reaction system was influenced by many different factors such as temperature and concentration of substrates. The ideal system contains protein 1.50 mg.mL-1, L-Arg 1 mmol.L-1, NADPH 0.1 mmol.L-1 at 37 degrees C. In this method, there were about 2% samples which emit fluorescence, and about 0.5% samples which quench the fluorescence. So these samples were deleted from the sample library. The effects of these samples on activity of NOS were distributed in a normal manner. About 2% samples had potential effects on the NOS activity (including inhibitors and enhancers).

CONCLUSIONThe method can be performed by high throughput screening and gives the stable data, not only for inhibitors, but also for enhancers of NOS activity.

Animals ; Arginine ; pharmacology ; Cattle ; Drug Evaluation, Preclinical ; methods ; Enzyme Inhibitors ; isolation & purification ; pharmacology ; In Vitro Techniques ; NADP ; pharmacology ; Nitric Oxide Synthase ; antagonists & inhibitors ; metabolism ; Nitroarginine ; pharmacology

Objective To investigate the source and genetic background of methicillin-resistant Staphylococcus aureus in the year of 2006,in China. Methods From January to December 2006,a total number of 302 consecutive and non-repetitive methicillin-resistant Staphylococcus aureus were collected from 17 Teaching hospitals in 15 areas. Genotypes of SCCmec were determined by multiplex PCR and multilocus sequence typing (MLST) was used to type the house-keeping genes. The implementation of the spa typing method was straightforward,and the results obtained were reproducible,unambiguous,and easily interpreted. Results All areas but Dalian harbored SCCmec Ⅲ while Dalian harbored SCCmec Ⅱ most. There were two strains in Guangzhou,harboring SCCmec Ⅳ. There were four strains of sequence type(ST),with ST239 accounted for 46.7% and ST5 accounted for 44.4%. ST59 accounted for 6.7% and ST88 accounted for 2.2%. There were fourteen strains of Spa typing,with t30 accounted for 52.6% ; t37 accounted for 27.2% ; t2 accounted for 12.9% ; t632 accounted for 2.3% ; t437 accounted for 1.3% ; t570,t601 accounted for 0.7% ; t377,t459,t796,t899,t1152,t2649 accounted for 0.3% ; no-typing accounted for 0.3%,respectively,pvl gene was not detected. Conclusion The main clone strains were ST239-MRSA-SCCmec Ⅲ-t30,ST5-MRSA-SCCmec Ⅱ-t2,with unique geographic distributions across the whole nation.

OBJECTIVETo investigate whether homocysteine (Hcy) participates the proliferation of the spontaneously hypertensive rat(SHR) vascular smooth muscle cell (VSMCs) and the molecular mechanism.

METHODSThe rat's arota were removed. The primary SHR VSMCs were isolated and cultured in vitro, then the SHR VSMCs were divided into four groups: (1) control group, (2) Hcy group, (3) 18alpha-glycyrrhetinic acid (GA) group, (4) Hcy + 18alpha-GA group. We detected proliferation of the SHR VSMCs by MTT and flow cytometry. The expression and co-localization of the connexin (Cx) 43 and Cx40 proteins in the SHR VSMCs were deteced by immunofluorescence. The expression of the Cx43 and Cx40 proteins in SHR VSMCs were detected by Western blot. The molecular dye transfer method (scrape dye transfer method) was applied to detect the gap junction function in the SHR VSMCs.

RESULTS(1) The Cx43 and Cx40 proteins expression in the SHR VSMCs were positive, confocal microscopy supported the co-localization of Cx43 and Cx40 in the cytoplasm. (2) The S value deteced by cell cycle and A value detected by MTT in the Hcy group were increased obviously compared with those in the control group (P < 0.05), decreased in 18alpha-GA group (P < 0.05). Compared with the Hcy group, the S and A value in the Hcy + 18alpha-GA group were significantly decreased, respectively (P < 0.05). (3) The expression of Cx43 and Cx40 proteins in Hcy group were increased compared with the control group (P < 0.05), decreased in 18alpha-GA group (P < 0.05). Compared with the Hcy group, the expression of Cx43 and Cx40 proteins in the Hcy + 18alpha-GA group were significantly decreased, respectively (P < 0.05). (4) The function of gap junction detected by scrape dye transfer method in the Hcy group were enhanced compared with the control group (P < 0.05), weakened in the 18alpha-GA group (P < 0.05). Compared with the Hcy group,the function of gap junction in the Hcy + 18alpha-GA group was significantly weakened (P < 0.05).

CONCLUSIONHcy can enhance the function of gap junctional to stimulate the proliferation of SHR VSMCs through the expression of Cx43 and Cx40 proteins promoted.

Animals ; Cell Proliferation ; Cells, Cultured ; Connexin 43 ; metabolism ; Connexins ; metabolism ; Gap Junctions ; metabolism ; Glycyrrhetinic Acid ; analogs & derivatives ; pharmacology ; Homocysteine ; pharmacology ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Rats ; Rats, Inbred SHR

BACKGROUNDMagnifying narrow-band imaging has enabled observation of the mucosal and vascular patterns of gastrointestinal lesions. This study investigated the potential value of magnifying endoscopy with narrow-band imaging for the classification of gastric intraepithelial neoplasia.

METHODSSeventy-six patients with gastric intraepithelial neoplasia (82 lesions) at People's Liberation Army General Hospital from December 2009 to November 2010 were analyzed. All patients underwent magnifying endoscopy with narrow-band imaging, and their lesions were differentiated into probable low-grade intraepithelial neoplasia or possible high-grade intraepithelial neoplasia on the basis of the imaging features. Pathologic proof was subsequently obtained by endoscopic submucosal dissection in every case. The validity of magnifying endoscopy with narrow-band imaging was calculated, considering histopathology to be the gold standard.

RESULTSMagnifying endoscopy with narrow-band imaging showed 22 low-grade intraepithelial neoplastic lesions and 60 high-grade intraepithelial neoplastic lesions. Of the 22 low-grade intraepithelial neoplastic lesions, 16 showed the same results on both imaging and pathology. Of the 60 high-grade intraepithelial neoplastic lesions, 53 showed the same results on both imaging and pathology. Thus, the sensitivity of magnifying endoscopy with narrow-band imaging for high-grade intraepithelial neoplasia was 89.83%, which was higher than that for low-grade intraepithelial neoplasia (69.57%). However, the specificity for high-grade intraepithelial neoplasia (69.57%) was lower than that for low-grade intraepithelial neoplasia (89.83%). The overall accuracy of magnifying endoscopy with narrow-band imaging was 84.15%.

CONCLUSIONSMagnifying endoscopy with narrow-band imaging can distinguish between gastric low- and high-grade intraepithelial neoplasia. It may be a convenient and effective method for the classification of gastric intraepithelial neoplasia.

Adult ; Aged ; Aged, 80 and over ; Carcinoma in Situ ; diagnosis ; Endoscopy ; methods ; Female ; Humans ; Male ; Middle Aged ; Stomach Neoplasms ; diagnosis

OBJECTIVETo explore the expression of integrin-linked kinase (ILK) and its effect on VEGF expression in fibroblasts from human hypertrophic scar.

METHODSFibroblasts were isolated from hypertrophic scar of 8 patients and cultured in vitro. Then the cells were divided into three groups: (1) Cells were cultured only in DMEM containing 10% FCS in the control group; (2) Cells were transfected with empty plasmid in the empty plasmid group; (3) Cells were transfected with plasmid expressing ILKcDNA in the ILK cDNA plasmid transfection group. First, the expression of ILK and VEGF was observed by immunocytochemistry before and after ILK cDNA transfection. Second, ILK and VEGF mRNA expression was investigated by real-time PCR (RT-PCR). Third, the protein expression of ILK and VEGF was detected by Western blot. Finally, the protein level of VEGF in supernatant of fibroblasts was measured by ELISA.

RESULTSBefore ILK cDNA transfection, the expression of ILK was positive and the VEGF expression was weak in cytoplasm of fibroblasts . After ILK cDNA transfection, both the expression of ILK and VEGF was enhanced. The level of VEGF mRNA was significantly higher in ILK cDNA transfection group (0.338 +/- 0.060) than that in control group (0.022 +/- 0.001) and empty plasmid group (0.028 +/- 0.005, P < 0.05). The level of VEGF protein was significantly higher in ILK cDNA transfection group (0.819 +/- 0.019) than that in control group (0.607 +/- 0.033) and empty plasmid group (0. 591 +/- 0.024, P<0. 05). Secretion of VEGF increased remarkably in ILK cDNA transfection group comparing with the other two groups (P < 0.05).

CONCLUSIONSILK could up-regulate the VEGF mRNA and protein level in human scar fibroblasts. It may play an important role in the angiogenesis in hypertrophic scar.

Cells, Cultured ; Cicatrix, Hypertrophic ; genetics ; metabolism ; pathology ; Fibroblasts ; secretion ; Humans ; Plasmids ; Protein-Serine-Threonine Kinases ; genetics ; RNA, Messenger ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo explore the expression of integrin-linked kinase (ILK) in scar in different growth stages, as well as its relationship with angiogenesis.

METHODS(1) Fifteen burn patients with scar formation time shorter than 6 months, ranging from 6 to 12 months, and longer than 12 months were hospitalized from December 2009 to December 2010. They were divided into A, B, and C groups according to the scar formation time, with 5 patients in each group. Scar specimens were harvested for observation of ILK expression with immunohistochemistry method, and ILK mRNA expression with real time fluorescence quantitative RT-PCR. (2) Microvascular endothelial cells (MEC) were isolated from scar tissue in A group and cultured in vitro, and then they were purified by immunomagnetic beads and identified with coagulation factor VIII marked by immunofluorescence (fibroblasts from human normal skin were used as control). The cultured cells in logarithmic growth phase were divided into control group (cultured with M131 medium containing microvascular growth supplement), transfection 1 group (transfected with empty plasmid), and transfection 2 group (transfected with ILK cDNA plasmid) according to the random number table. After 24 hours, the expressions of ILK mRNA, Flt-1 mRNA, and KDR mRNA were determined with real time fluorescence quantitative RT-PCR. Data were processed with one-way analysis of variance.

RESULTSImmunohistochemical observation showed that ILK in A group mainly expressed in the basal layer cells of epidermis, cytoplasm of fibroblasts, and MEC in scar, while ILK in B group only distributed in the basal layer cells of epidermis, but ILK expression in C group was not obvious. The expression of ILK mRNA in A group (0.34 ± 0.16) was significantly higher than those in B and C groups (0.17 ± 0.06, 0.07 ± 0.13, F = 37.007, P = 0.000). MEC grew up showing cobble stone formation after purification. The expression of coagulation factor VIII was positive in cytoplasm of purified MEC, while that was negative in fibroblast of human normal skin. The expressions of ILK mRNA (57.807 ± 5.556), KDR mRNA (0.836 ± 0.014), and Flt-1 mRNA (0.162 ± 0.005) in transfection 2 group were higher than those in control and transfection 1 groups (0.018 ± 0.003, 0.028 ± 0.020, 0.023 ± 0.004 and 0.042 ± 0.005, 0.039 ± 0.007, 0.046 ± 0.003; F(ILK) = 87.110, F(KDR) = 11.241, F(Flt) = 18.199, with P values all below 0.01).

CONCLUSIONSILK mainly expressed in scar tissue with formation time shorter than 6 months, and it may affect vascularization of scar by regulating gene expressions of KDR and Flt-1 in MEC, which plays an important role in early scar formation.

Adolescent ; Adult ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Endothelial Cells ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Neovascularization, Pathologic ; metabolism ; pathology ; Protein-Serine-Threonine Kinases ; metabolism ; Vascular Endothelial Growth Factor Receptor-1 ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism ; Young Adult