OBJECTIVETo investigate whether RhoA/Rho-kinase contributes to the occurrence of chronic post-thoracotomy pain (CPSP) by up regulation of glutaminase 1 (GLS1) expression in the spinal dorsal cord.

METHODSTwenty five male Sprague Dawley (SD) rats were divided into control group (n=5) and model group (n=20). The rats in the model group were randomized into two sub groups (n=10) for observation on day 10 and day 21 after thoracotomy, and each group was further divided into CPSP and non CPSP groups according to the behavioral test results. All the rats were sacrificed after behavioral test for examination of GLS1 and RhoA expressions in the spinal cord using Western blotting and RT PCR. We also compared the effect of the Rho kinase inhibitor fasudil and saline, both injected intraperitoneally daily at 10 mg/kg for 7 consecutive days following thoracotomy, on CPSP and GLS1 expression in 30 male SD rats on day 21 after thoracotomy.

RESULTSCompared with the control group, the rats with CPSP showed significantly increased expressions of GLS1 and RhoA mRNA in the spinal cord on both day 10 and day 21 following thoracotomy (P<0.01), but the rats without CPSP did not show obvious changes in GLS1 and RhoA expressions. In fasudil treated rats, the mechanical pain threshold was obviously increased and the expressions of GLS1 and RhoA were significantly reduced as compared with those in saline treated rats (P<0.01).

CONCLUSIONRhoA plays an important role in the occurence of CPSP by up-regulating the expression of GLS1 in the spinal dorsal cord of rats.

OBJECTIVETo investigate the expressions of vascular endothelial growth factors (VEGF)-A, -C and -D and their prognostic significance and relation to angio- and lymphangiogenesis in gastric cancer.

METHODSThe expression of VEGF-A, -C and -D in 123 primary gastric cancers was detected by immunohistochemical staining. The lymphatic vessel density (LVD) and microvessel density (MVD) were assessed after immunohistochemical double-staining with D2-40 and CD34, respectively. The correlation between the expression of those VEGF factors and clinicopathological parameters were analyzed by univariate method. The overall survival was evaluated by Kaplan-Meier method and log-rank test. Multivariate analysis was carried out using Cox proportion hazard model.

RESULTSThe positive expression rate of VEGF-A, -C and -D in primary gastric cancer samples were 64.2%, 65.9% and 41.5%, respectively. High expression of VEGF-A, or -C or -D, or any two of them was correlated with high LVD (P < 0.05). High expression of both VEGF-A and -C was associated with high MVD, lymph node metastasis, LVI and MVI (P < 0.05). Both VEGF-C and -D high expression was correlated with LVI and lymph node metastasis (P < 0.05). The patients with high expression of these factors had a statistically shorter overall survival (P < 0.05). The patients with both VEGF-A and -C expression had the shortest survival (56 months). Multivariate analysis showed that VEGF-A high expression, MVD, lymph node metastasis and depth of tumor invasion were independent survival predictors (P = 0.033, 0.002, 0.019 and P < 0.001, respectively).

CONCLUSIONHigh expression of both VEGF-A and -C imply high potential of lymphangiogenesis, metastasis and poorer survival in gastric cancer patients. High expression of VEGF-C and -D may induce lymphangiogenesis and promote lymph node metastasis, but only VEGF-A is an independent predictor of survival.

Adult ; Aged ; Aged, 80 and over ; Female ; Follow-Up Studies ; Humans ; Lymphangiogenesis ; Lymphatic Metastasis ; Lymphatic Vessels ; pathology ; Male ; Microvessels ; pathology ; Middle Aged ; Neovascularization, Pathologic ; Proportional Hazards Models ; Stomach Neoplasms ; metabolism ; pathology ; Survival Rate ; Vascular Endothelial Growth Factor A ; metabolism ; Vascular Endothelial Growth Factor C ; metabolism ; Vascular Endothelial Growth Factor D ; metabolism

OBJECTIVETo investigate the susceptibility of Streptococcus mutans (S. mutans) biofilms to antimicrobial agent by confocal laser scanning microscopy (CLSM).

METHODSS. mutans biofilms formed in vitro on glass slice were acted on with penicillin of different concentrations for 3 h. Then these biofilms were stained by fluorescence and were observed by CLSM. The bacterial density and viability of biofilms were recorded.

RESULTSWhen S. mutans biofilms were exposed to penicillin of 2 500 mg/L for 3 h, it was not completely killed. The higher the concentration of penicillin was, the weaker the biofilms against penicillin.

CONCLUSIONSCompared with planktonic S. mutans, S. mutans biofilms produced stronger resistance to penicillin. It suggests that we should find new strategies to control the infection caused by biofilm in clinic.

Anti-Infective Agents ; administration & dosage ; pharmacology ; Biofilms ; drug effects ; Dose-Response Relationship, Drug ; Microbial Sensitivity Tests ; Microscopy, Confocal ; Penicillins ; administration & dosage ; pharmacology ; Streptococcus mutans ; drug effects

BACKGROUNDTo investigate the frequency of circulating HBV specific T helper cell and evaluate its association with serum levels of HBV DNA before and during lamivudine treatment in patients with chronic hepatitis B.

METHODSThe frequency of circulating HBV specific T helper cells in response to HBcAg in 25 chronic HBV-infected patients was determined by Elispot assay; serum HBV DNA was quantitated by real-time PCR.

RESULTSThe frequency of HBV specific T helper cell before antiviral treatment (47.30 +/- 25.50 SFCs /1 x 10(6) PBMC) was significantly higher than that at the third month of therapy (23.10 +/- 18.45 SFCs /1 x 10(6) PBMC, P < 0.05). All 8 patients observed dynamically had decreased frequency of HBV specific T helper cell at the third month of therapy; six patients with serum HBV DNA level reduced had higher frequency of HBV specific T helper cell before treatment than 2 patients without serum HBV DNA level decrease.

CONCLUSIONHBV specific T helper cell response at the time of hepatitis flare in chronic hepatitis B patients was significantly augmented compared to that at the time of catabasis.

Adult ; Antiviral Agents ; therapeutic use ; DNA, Viral ; blood ; genetics ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Hepatitis B Core Antigens ; immunology ; Hepatitis B virus ; drug effects ; genetics ; immunology ; Hepatitis B, Chronic ; blood ; drug therapy ; virology ; Humans ; Lamivudine ; therapeutic use ; Male ; T-Lymphocytes, Helper-Inducer ; cytology ; drug effects ; immunology

AIMThe purpose of this study was to develop a mathematical model to quantitatively describe the passive transport of macromolecules within dental biofilms.

METHODOLOGYFluorescently labeled dextrans with different molecular mass (3 kD, 10 kD, 40 kD, 70 kD, 2000 kD) were used as a series of diffusion probes. Streptococcus mutans, Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum were used as inocula for biofilm formation. The diffusion processes of different probes through the in vitro biofilm were recorded with a confocal laser microscope.

RESULTSMathematical function of biofilm penetration was constructed on the basis of the inverse problem method. Based on this function, not only the relationship between average concentration of steady-state and molecule weights can be analyzed, but also that between penetrative time and molecule weights.

CONCLUSIONThis can be used to predict the effective concentration and the penetrative time of anti-biofilm medicines that can diffuse through oral biofilm. Furthermore, an improved model for large molecule is proposed by considering the exchange time at the upper boundary of the dental biofilm.

Actinomyces ; growth & development ; Algorithms ; Biofilms ; growth & development ; Biological Transport ; Dental Plaque ; microbiology ; Dextrans ; pharmacokinetics ; Diffusion ; Fluorescent Dyes ; pharmacokinetics ; Fusobacterium nucleatum ; growth & development ; Macromolecular Substances ; pharmacokinetics ; Microscopy, Confocal ; Models, Biological ; Molecular Probe Techniques ; Streptococcus mutans ; growth & development ; Streptococcus sanguis ; growth & development

Stem cell growth factor (SCGF) is an early-acting hematopoitic cytokine that has two isoforms including hSCGF with full length molecules and hSCGFbeta, 78 amino acids of which lost in the conserved calcium-dependent carbohydrate-recognition domain (CRD). It has been demonstrated that hSCGFbeta is strictly species-specific in regulating he-matopoiesis. This study was aimed to explore whether human SCGF can exert synergistic stimulatory effect on heterogenous murine CFU-GM progenitor. Firstly, hSCGF cDNA was amplified from human fetal liver cDNA library by using two-step PCR. The hSCGF mature peptide coding sequence was subsequently placed at downstream of glutathione S-transferase (GST) sequence in GST gene fusion expression vector. The results indicated that there existed an additional 60 kD protein compared with mock BL21 when the cells hosting recombinant plasmid were induced with IPTG at 37 degrees C. SDS-PAGE analysis demonstrated that the GST-hSCGF fusion protein mainly existed in insoluble form. When induced at low temperature (28 degrees C), the recombinant protein was mostly soluble. The GST-fusion recombinant protein was subsequently purified by using affinity chromatography. The clonogenic assay revealed that, unlike hSCGFbeta, hSCGF had the granulocyte/macrophage promoting activity (GPA) for murine bone marrow GM progenitor. It is concluded that, in contrast to human SCGFbeta, the intact molecular hSCGF may have no species specificity, implying that CRD domain in human SCGFbeta does not directly bind to corresponding SCGF receptor, but may have certain biological function.
Cloning, Molecular ; DNA, Complementary ; biosynthesis ; genetics ; isolation & purification ; Hematopoiesis ; genetics ; Humans ; Species Specificity ; Stem Cell Factor ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo construct a high metastatic potential human hepatocellular carcinoma (HCC) orthotopic transplantation model with palliative liver resection in nude mice.

METHODSA human HCC orthotopic nude mice model was established by administering a single inoculation of the highly metastatic MHCC97H tumor tissue (size 2 mm * 2 mm * 2 mm) into the left liver lobe. At day 14 post-inoculation, a random group of the mice received palliative liver resection; the unresected mice served as controls. Changes in expression levels of 113 genes with metastasis-related functions were evaluated in the residual HCC tissues. At day 35 post-resection, a random group of the mice were sacrificed by cervical dislocation and a comprehensive metastases examination was performed. The remaining mice were used to observe life span. All statistical analyses were performed by the SPSS v17.0 software, and significance was defined as P less than 0.05.

RESULTSThe nude mouse model of highly metastatic HCC with palliative liver resection was successfully established. Incidences of intrahepatic and abdominal metastases were higher in the palliative resected group (vs. unresected group: 11.7+/-4.7 vs. 6.3+/-2.8, t = -2.412, P less than 0.05 and 9.8+/-3.4 vs. 5.2+/-2.6, t = -2.641, P less than 0.05 respectively). In addition, the palliative resected group showed significantly enhanced pulmonary metastasis (vs. unresected group: 14.3+/-4.7 vs. 8.7+/-4.7, t = -2.348, P less than 0.05). Differential gene expression levels were found for MTSS1, TGFbl, SMAD2, IL-1b, and MMP7, and were situated in the central position of gene function net of residual HCC. The life-span of the palliative resected group was significantly longer than that of the unresected group (60.8+/-2.7 vs. 51.3+/-1.4 days, x2 = 12.850, P less than 0.01).

CONCLUSIONThe highly metastatic human HCC nude mouse model with palliative liver resection that was successfully constructed in this study represents a useful investigational tool to assess the biological characteristics of residual cancer and to screen therapeutic strategies.

Animals ; Carcinoma, Hepatocellular ; pathology ; surgery ; Disease Models, Animal ; Hepatectomy ; Humans ; Liver Neoplasms, Experimental ; pathology ; surgery ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Metastasis ; Neoplasm Transplantation ; Tumor Cells, Cultured

OBJECTIVETo compare the inhibitory effects of cytokine-induced killer (CIK) cells alone, chemotherapeutic drug alone, and CIK cells combined with chemotherapeutic drug on the growth of hepatocellular carcinoma (HCC) cells transplanted in nude mice.

METHODSPeripheral blood mononuclear cells (PBMC) collected from five healthy donors by blood cell separator were incubated in vitro to induce CIK cells in the presence of interferon-gamma (IFN-gamma), IL-2 and anti-CD3 monoclonal antibody (mAb). The phenotype of CIK cells was characterized by flow cytometric analysis. BEL-7402 HCC cells were inoculated subcutaneously to nude mice. On day 5, at the inoculation site were injected normal saline (group 1), CIK cells (3 x 10(7) and 6 x 10(7), group 2 and 3), mitomycin-C (MMC 80 microg in 0.2 ml, group 4), and CIK cells combined with MMC (group 5), respectively.

RESULTSThe percentage of CD3(+), CD3(+)CD8(+), CD3(+)CD56(+), CD25(+) cells increased from 64.0%, 28.0%, 7.8%, and 9.1% to 94.7%, 67.7%, 61.3%, and 84.0% respectively after cytokine induction. The percentage of CD3(+) and CD3(+)CD8(+) cells remained at high levels during incubation period, but that of CD25(+) and CD3(+)CD56(+) cells peaked respectively on day 7 and 13 and then declined. During the 90-day observation, the tumor formation rates were 100%, 70.0%, 80.0%, 70.0% and 66.7%; and the mouse survival rates were 10.0%, 60.0%, 40.0%, 50.0% and 75.0%, respectively from group 1 to group 5. Compared to the other groups, in the combined therapy group of mice, not only the tumor grew slowly and but also showed more marked tissue necrosis.

CONCLUSIONThe growth inhibitory effect on human HCC transplanted in nude mice of combined CIK cells and MMC treatment is more potent than that of CIK cells or MMC alone.

Animals ; Antibiotics, Antineoplastic ; therapeutic use ; Carcinoma, Hepatocellular ; immunology ; pathology ; therapy ; Cell Line, Tumor ; Cells, Cultured ; Combined Modality Therapy ; Cytokines ; metabolism ; pharmacology ; Female ; Humans ; Immunotherapy, Adoptive ; Killer Cells, Natural ; transplantation ; Liver Neoplasms ; immunology ; pathology ; therapy ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Mitomycin ; therapeutic use ; Neoplasm Transplantation

OBJECTIVETo define the anatomical approach, anatomical planes and related vessels and nerves to create a safe and reproducible combined sublingual and bi-vestibular access for trans-oral video-assisted thyroidectomy.

METHODSFrom November 2009 to May 2011, twenty-five embalmed human specimens were dissected for anatomical information of the cervical region, the mandible region and the supra-hyoid muscles. On twenty fresh frozen human specimens after an experimental trans-oral endoscopic thyroidectomy, the related vascular, neural structures and muscles were evaluated.

RESULTSThe optical access port was placed in the midline sublingual. The geniohyoid muscle, mylohyoid muscle and the anterior belly of the digastric muscle were divided in the midline in order to reach the plane under the platysma muscle. The mucosa was sagittal incised bilaterally in the vestibular of oral cavity for working trocar, at the level of the first molar of the mandible. The working trocar reached directly the periosteum of the mandible, under the facial vessel and the marginal branch of facial nerve, and then passed below the platysma muscle into the infra-laryngeal working area. The distance from mental nerve to mandibular midline and between mental nerve and facial artery were (25.8 ± 0.9) mm and (29.4 ± 0.9) mm respectively. Anatomical dissections showed that after an experimental trans-oral combined sublingual and bi-vestibular access, all muscles of the floor of the oral cavity as well as the related vascular and neural structures are intact. The maximum nodule size of the resected specimens in the totally trans-oral approach was up to 50 mm.

CONCLUSIONThe combined sublingual and bi-vestibular access of trans-oral video-assisted thyroidectomy is safe and reproducible.

Adolescent ; Adult ; Female ; Humans ; Male ; Mandible ; anatomy & histology ; Middle Aged ; Mouth ; anatomy & histology ; Mouth Floor ; anatomy & histology ; Thyroidectomy ; methods ; Young Adult

OBJECTIVETo investigate the effects of lentivirus mediated siRNA targeting human metastasis suppressor 1 (MTSS1, MIM-B gene) gene on the invasive and metastatic potentials of hepatocellular carcinoma (HCC) MHCC97H cells.

METHODSThe siRNA targeting MTSS1 was cloned into one lentivirus work vector. The work vector and three package plasmids were co-transfected into 293T cells with the help of lipefeetamine 2000. Lentivirus was collected in 72 hours and was added to the cultured MHCC97H cells. The total cell MIM-B mRNA and MIM-B protein were extracted and underwent real-time PCR and western-blot test respectively. Boden chamber assay was used to evaluate the invasive potential of MHCC97H cells. Gelatin zymography was used to detect matrix metalloproteinase-2 (MMP2) activity. Metastatic human HCC nude mice models were established by orthotopic implantation with a high metastatic potential human HCC cell line MHCC97H. Twenty-four nude mice bearing orthotopic xenografts were randomized into black control group, Lenti-GFP group and intervention group (Lenti-MTSS1 group) 14 days after orthotopic implantation (8 per group). The ultrasound-guided multi-point injection was performed on mice with borate buffered saline, Lenti-GFP and Lenti-MTSS1 respectively. Mice were sacrificed on day 35 for the examination of pulmonary metastasis. The SPSS 13.0 soft ware was applied to data analysis.

RESULTSThe small interfering RNA targeting MTSS1 was constructed successfully with a transfection efficiency of 97.0%, which produced a marked inhibition of invasive ability of MHCC97H cells through Matrigel, being 37.9+/-4.4, 37.4+/-5.3 and 26.6+/-4.6 in the black control group, Lenti-GFP group and Lenti-MTSS1 group (F = 26.695, P value is less than 0.01), respectively. MIM-B expression and MMP2 activity of intervention group were also significantly down-regulated as compared to the control group. The results of in vivo studies showed that the numbers of lung metastatic nodules were 6.5+/-2.6, 6.4+/-2.7 and 3.8+/-1.3 in the black control group, Lenti-GFP group and intervention group respectively with significant statistical difference (F = 3.637, P value is less than 0.05), accorded with tumor tissue MIM-B mRNA expression of 0.39+/-0.19, 0.38+/-0.10 and 0.16+/-0.11 respectively (F = 11.644, P value is less than 0.01) when comparison was made between control group and therapy group.

CONCLUSIONSmall interfering RNA mediated by lentivirus inhibited MIM-B expression and resulted in inhibition of the invasive and metastatic potentials of MHCC97H cells, which may attributed, in part, the down regulation of MMP2 activity, and thus may provide a new molecular targeted therapy for HCC patients in the future.

Animals ; Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Line, Tumor ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microfilament Proteins ; genetics ; Neoplasm Proteins ; genetics ; Neoplasm Transplantation ; RNA, Small Interfering ; genetics ; Transfection