Animals ; Hepatocyte Growth Factor ; metabolism ; Humans ; Proto-Oncogene Proteins c-met ; Serine Endopeptidases

OBJECTIVETo investigate the role of activated hepatocyte growth factor (HGF) in apoptosis of hepatic stellate cells (HSCs) and in modulating the Rho signaling pathway.

METHODSHSCs were divided into the following groups: blank control, consisting of HSCs without treatment; two treatment controls, consisting of HSCs exposed to exogenous HGF at 50 ng/ml and HSCs exposed to exogenous HGF activator (HGFA) at 70 ng/ml; three experimental groups, consisting of HSCs exposed to both exogenous HGF and HGFA, HSCs pre-incubated with the HGF inhibitor c-met at 500 ng/ml for 6 hours and then exposed to exogenous HGF and HGFA, and HSCs pre-incubated with the Rho pathway inhibitor Y-27632 at 10 ng/ml and then exposed to exogenous HGF and HGFA. Activation status of the cultured HSCs was determined by change in expression of alpha-smooth muscle actin (SMA). The optimal intervention concentration of Y-27632 was determined by MTT assay. The apoptotic status of HSCs was determined by flow cytometry. Expression of the HGF-alpha chain was detected by immunofluorescence. The expression of RhoA was evaluated by PCR (for mRNA) and by immunohistochemical staining and Western blot analysis (for protein).

RESULTSExposure to 10 mumol/L Y-27632 led to obvious growth inhibition of HGF + HGFA-induced HSCs, compared with the other concentrations tested (P less than 0.05). HGF + HGFA induced the expression of the HGF-alpha chain in a time-dependent manner (P less than 0.01); however, the increases in expression of HGF-alpha chain induced by HGF alone and HGFA alone were not significantly different from the level in the blank controls (P more than 0.05). Exposure to HGF alone and HGFA alone led to a time-dependent increase in apoptosis (24 h, 48 h, 72 h) but exposure to HGF + HGFA led to the highest levels of apoptosis (P less than 0.05). Exposure to HGF + HGFA led to a time-dependent decrease in RhoA mRNA and protein expression (P less than 0.01).

CONCLUSIONActivation of hepatocyte growth factor promotes apoptosis of hepatic stellate cells by suppressing RhoA expression and down-regulating the Rho signaling pathway.

OBJECTIVETo compare agitated saline solution (AS) and the mixture of AS with blood (ASb) as the contrast agents in contrast transcranial Doppler (c-TCD) in the diagnosis of patent foramen ovale (PFO).

METHODSWe recruited 248 consecutive patients for c-TCD examination between November 2015 and January 2016, and the sequence of the use of AS (9 mL saline solution mixed with 1 mL air) and ASb (9 mL saline solution and a drop of the patient's blood mixed with 1 mL air) was determined by coin-tossing method. Before the examination, the contrast agent was injected with or without Valsalva maneuvers (VM), and the number of microbubbles within 25 s after the contrast agent injection and the time of first appearance of microbubbles were recorded by observing the TCD spectrum. Each injection was repeated twice and the interval between tests was at least 5 min. We classified PFO according to the number of microbubbles into negative (no microbubble), grade I (1-10 microbubbles), grade II (>10 microbubbles but no curtain), and grade III (with curtain).

RESULTSs The positivity rates in diagnosis with AS without VM, AS with VM, ASb without VM, and ASb with VM tests were 10.9%, 23.8%, 12.1% and 25.8%, respectively. AS with VM test had a higher positive rate than AS without VM test (23.8% vs 10.9%, P=0.001), and ASb with VM test had a higher positive rate than ASb without VM test (25.8% vs 12.1%, P=0.001). The positive rates were similar between ASb without VM and AS without VM test (12.1% vs 10.9%, P=0.250) and between ASb with VM test and AS with VM test (25.8% vs 23.8%, P=0.125).

CONCLUSIONVM can improve the positive rate of PFO diagnosis in c-TCD examination, and the positive rates are comparable between examinations using the contrast agents AS and ASb.

Contrast Media ; chemistry ; Foramen Ovale, Patent ; diagnostic imaging ; Microbubbles ; Sensitivity and Specificity ; Sodium Chloride ; Ultrasonography, Doppler, Transcranial ; Valsalva Maneuver

OBJECTIVETo observe the clinical effect of a occipitocervical fixation systems using C(2) pedicle screws and plates.

METHODSAn occipitocervical fixation system was designed. Since June 2001 to March 2003, 38 patients with instability of atlantoaxial joint underwent reconstructive surgery using this systems. Twenty-four patients were associated with congenital occipitalization. The pedicle screws were inserted into C(2) pedicles in the direction as its axis. The occipitocervical plate was slightly bent to fit the occipital contour and fixed onto the occiput. Hyperflexion alignment of the occipitoatlantoaxial complex was corrected by application of extensional force created by tightening of the nut on the pedicle screws. The autogenous cancellous bones were grafted between the occiput and the axis.

RESULTSIn this series, neither vertebral artery nor spinal cord was injured. 36 of 38 cases were followed up for an average of 18 months, all cases achieved solid bony fusion. No implant failure was found.

CONCLUSIONSOccipitocervical reconstruction by the combination of C(2) pedicle screws and occipitocervical plate systems can provide sufficient correction of malalignment in the occipitoatlantoaxial region and achieve high fusion rate.

Adolescent ; Adult ; Atlanto-Axial Joint ; surgery ; Bone Plates ; Bone Screws ; Cervical Vertebrae ; surgery ; Child ; Female ; Follow-Up Studies ; Humans ; Joint Instability ; surgery ; Male ; Middle Aged ; Occipital Bone ; surgery ; Reconstructive Surgical Procedures ; methods ; Spinal Fusion ; instrumentation ; methods ; Treatment Outcome

OBJECTIVETo compare the values of dual-source computed tomographic angiography (DSCTA) and digital subtraction angiography (DSA) in evaluating intracranial aneurysms in patients with nontraumatic subarachnoid hemorrhages (SAH) .

METHODSTotally 80 nontraumatic SAH patients were evaluated with both DSCTA and DSA. With the results of DSA as the gold standards, the sensitivity, specificity, and accuracy of DSCTA was calculated, and the agreement between these two modes was analyzed.

RESULTSA total of 73 aneurysms were detected by DSCTA in 65 patients, 74 aneurysms were detected by DSA in 63 patients. 2 cases were false positive and 3 aneurysms were missed by DSCTA. The sensitivity, specificity and accuracy of DSCTA were 95.5%, 88.2%, and 94% on a per-patient basis and were 95.9%, 88.2%, and 94.5% on a per-aneurysm basis.

CONCLUSIONFor patients with nontraumatic SAH, DSCTA and DSA have comparable abilities in detecting and displaying aneurysms.

Adolescent ; Adult ; Aged ; Angiography, Digital Subtraction ; Cerebral Angiography ; methods ; Female ; Humans ; Intracranial Aneurysm ; diagnostic imaging ; Male ; Middle Aged ; Sensitivity and Specificity ; Tomography, X-Ray Computed ; Young Adult

OBJECTIVETo investigate the specific cytotoxity of tumor infiltrating lymphocytes (TIL) transfected with chimeric T cell receptor (CTCR) on cells which express KDR.

METHODSA recombinant retroviral plasmid (pMSCVneo-Vhgamma) was constructed by cloning VEGF121-hinger-FcRgamma (Vhgamma) into retroviral vector pMSCVneo. After packaging by PT67, the virus with high titer was used to infect TIL isolated from liver cancer tissues, and then MSCVneo-Vhgamma-TIL was generated. TIL infected with MSCVneo was used as a control. The cytotoxicty of the transgenic TIL on NIH3T3 and HepG2 expressing no KDR and on ECV304 and A375 expressing KDR was detected with MTT colorimetric assay.

RESULTSThe sequences of VEGF121 and hinger-FcRgamma were different from those reported, but the deduced amino sequences were identical to the reported ones. The cytotoxity of TIL infected with MSCVneo on target cell was similar to that of the control TIL; both only had mild cytotoxity on cancer cell line. No significant cytotoxity was found in TIL infected with MSCVneo-cTCR on NIH3T3, but its cytotoxity on ECV304 was significant. The cytotoxity on HepG2 was similar to that of MSCVneo-TIL and uninfected TIL, but cytotoxity on A375 was significantly higher.

CONCLUSIONChimeric T cell receptor permanently grafts TIL cell with predefined new specificity. TIL expressing Vhgamma can selectively recognize and kill vascular endothelial cell and tumor cells which express VEGF receptor KDR.

Animals ; Cytotoxicity, Immunologic ; Humans ; Lymphocytes, Tumor-Infiltrating ; immunology ; Mice ; NIH 3T3 Cells ; Plasmids ; Receptors, Antigen, T-Cell ; physiology ; Retroviridae ; genetics ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; physiology

OBJECTIVETo investigate the relationship between serum alpha-fetoprotein (AFP) variant levels in patients with hepatocellular carcinoma (HCC) and cancer cells disseminating through blood.

METHODSSerum AFP variant levels were measured by crossed immunoaffino-electrophoresis in the presence of lectin before initial surgical treatment in HCC patients. Circulating tumor cells were simultaneously detected in pre-operative blood samples using reverse transcription-polymerase chain reaction (RT-PCR) for AFP mRNA.

RESULTSForty-six HCC patients with serum AFP positive were studied. Serum AFP variant level > or 20% was showed in 37 patients, among whom there were 22 (59.5%) showing AFP mRNA positive. In contrast, the positive AFP mRNA expression was only observed in 2 out of 9 patients (22.2%) with AFP variant level<20% (x(2)=4.02, P<0.05).

CONCLUSIONIn hepatocellular carcinoma patients, increased AFP variant levels are associated with a haematogenous spread of tumor cells.

Adult ; Aged ; Carcinoma, Hepatocellular ; blood ; Female ; Humans ; Liver Neoplasms ; blood ; Male ; Middle Aged ; RNA, Messenger ; blood ; Reverse Transcriptase Polymerase Chain Reaction ; alpha-Fetoproteins ; analysis ; genetics

OBJECTIVEThe scientific basis and the clinical effectiveness of allergen specific immunotherapy (SIT) administered by subcutaneous injection are well established. This study aimed to observe the changes in amount of inhaled corticosteroids, total IgE, specific IgE, peak expiratory flow rate (PEF), etc. during a standardized SIT against house dust mite in allergic asthmatic children.

METHODChildren (5 - 13 years old) with mild to moderate allergic asthma seen from February 2005 to June 2008 were enrolled into this study. A non- randomized retrospective study was performed. All children were diagnosed sensitive to dust mites, the treatment group accepted standardized dust mite allergen specific immunotherapy. Each fourth injections were defined as observation points, the study took 3.4 years. The investigators recorded the treatment, the cumulative allergen extract, changes of daily doses of inhaled corticosteroid, peak expiratory flow (PEF), total IgE (TIgE), specific IgE (SIgE). The control group only received inhaled corticosteroids. The daily doses of inhaled corticosteroid and the number of asthma attacks, and the control rate were compared between the 2 groups.

RESULTTotally 85 children were treated with SIT [(7.6 ± 1.4) years], 45 males and 40 females; 50 children received only drug treatment [(7.7 ± 1.5) years], 28 males and 22 females. The cumulative dose of allergen was up to (69.7 ± 4.8) µg after the 20 times injection, the dose of inhaled corticosteroids was significantly less than that in the control group (t = 2.359, P < 0.05). PEF was significantly higher than that of pre-treatment level (F = 7.874, P < 0.05). TIgE and SIgE had no significant change (t = 0.313, P > 0.05, t(Derp) = 0.517, t(Derf) = 0.717, P > 0.05). After the treatment, the control rate of the SIT group was 85.5%, that of the control group was 62.0% (χ(2) = 10.150, P < 0.01).

CONCLUSIONThe standardized SIT against house dust mite could reduce steroid use in mild to moderate allergic asthmatic children. After (38.7 ± 2.3) weeks, the cumulative dose of allergen was up to (69.7 ± 4.8) µg, inhaled corticosteroid was significantly reduced. At the end of SIT, 85% of patients obtained complete control of asthma. Total IgE and mite-specific IgE had no significant changes.

Adolescent ; Animals ; Antigens, Dermatophagoides ; therapeutic use ; Asthma ; immunology ; therapy ; Child ; Child, Preschool ; Desensitization, Immunologic ; methods ; Female ; Glucocorticoids ; therapeutic use ; Humans ; Immunoglobulin E ; immunology ; Male ; Pyroglyphidae ; immunology ; Retrospective Studies ; Treatment Outcome

OBJECTIVETo determine whether apoptosis is induced in rat hepatic stellate cells (HSCs) in response to activation of the hepatocyte growth factor (HGF) by hepatocyte growth factor activator (HGFA) by using a co-culture system of bone marrow mesenchymal stem cells (BMSCs) and HSCs.

METHODSIn this study, cells were divided into the following five groups: HSC control group: HSCs co-cultured with fibroblast cells; HSCs blank group: HSCs cultured alone; BMSCs blank group: BMSCs cultured alone; Experimental group: BMSCs + HSCs; HGFA intervention group: HSCs treated with 70 ng/mL of HGFA. The culture systems were established in culture plates with transwell inserts, and cells were assessed at 24, 48, and 72 h of growth. Dynamic changes in cell morphology were observed under an inverted phase contrast microscope. The surface markers of BMSCs and the apoptosis rate of HSCs were detected by Annexin-V-FITC/propidium iodide (PI). Expression of a-smooth muscle actin (SMA) in HSCs was evaluated by immunohistochemistry. The presence of activated HGF (HGF-a chain) was determined by immunofluorescent staining. HSC proliferation was measured by MTT assay, and the concentrations of HGF and HGFA were quantified by enzyme-linked immunosorbent assay (ELISA).

RESULTSMTT results indicated that treatment with HGF alone had no effect on HSC proliferation rate (vs. HSC blank group, P more than 0.05), but that 24 h treatment with HGFA significantly inhibited the proliferation rate (0.26 ± 0.00 vs. blank group: 0.13 ± 0.04, P = 0.02); moreover, this effect was concentration-dependent. Expression of HGF-a was lower in the experimental group than in the HGFA intervention group at 72 h (37.24 ± 1.03 vs. 40.44 ± 0.77, P = 0.04), and both of these groups had higher expression than the control group at all time points examined (P less than 0.05). The apoptosis rate was consistently higher in the experimental group than in the HGFA intervention group, but most robustly at 72 h (40.77 ± 1.16% vs. 33.35 ± 2.04%, P = 0.00); moreover, the apoptosis rate was significantly higher than that in the control group at all time points examined (P less than 0.01). The concentration of HGF in the experimental group and the HGFA intervention group showed a time-dependent reduction, and was consistently lower than that in the HSCs control group (P less than 0.05). Finally, the concentration of HGFA was higher in the experimental group than in the blank group at all time points examined (P less than 0.05).

CONCLUSIONThe BMSC-HSC co-culture system can promote secretion of HGFA from HSCs and HGF activation, thereby inducing apoptosis of HSCs.

Animals ; Apoptosis ; drug effects ; Bone Marrow Cells ; cytology ; Cells, Cultured ; Coculture Techniques ; Hepatic Stellate Cells ; cytology ; Hepatocyte Growth Factor ; metabolism ; pharmacology ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley ; Serine Endopeptidases ; metabolism

OBJECTIVETo observe the effect of Tongsaimai (TSM) tablets in treating foot trauma of diabetic foot (DF) model rats, and discuss its potential mechanism.

METHODMale SD rats were selected to duplicate the diabetic foot ulcer model and randomly divided into the blank control group, the model group, the metformin treatment group, and TSM 12.44, 6.22, 3.11 g x kg(-1) groups (n = 10). The healing of ulcer wounds were observed on day 1, 4, 8, 13 and 18. After 18 days, a histopathologic examination was conducted for ulcer tissues. The contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by hydroxylamine and TBA methods. The content of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were determined with the radioimmunoassay. The immunohistochemical method was used to observe the expression of vascular endothelial growth factor (VEGF) in ulcer tissues and the number of capillary vessels.

RESULTTSM could alleviate the pathological changes of diabetic foot rats, accelerate the ulcer healing on 4, 8, 13, 18 d, reduce MDA, IL-6, TNF-alpha, VEGF content in rat serum at 18 d (after the rehabilitation period), and enhance the SOD content. Specifically, the TSM 12.44 g x kg(-1) group showed significant differences compared with the model group (P < 0.05, P < 0.01). At 18 d after the treatment (the late rehabilitation period), the VEGF expression of TSM 12.44, 6.22 g x kg(-1) groups and the number of blood capillaries of the TSM 12.44 g x kg(-1) group were significantly lower than that of the model group (P < 0.05, P < 0.01).

CONCLUSIONTSM could promote the foot wound healing of DF model rats, reduce MDA, IL-6 and TNF-alpha levels in serum, increase the SOD content and decrease the VEGF expression and the number of blood capillaries in the late rehabilitation period. Its action mechanism may be related to the inhibition of oxidative stress injury and the inflammatory cell infiltration.

Animals ; Diabetic Foot ; drug therapy ; genetics ; metabolism ; physiopathology ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Interleukin-6 ; genetics ; metabolism ; Male ; Malondialdehyde ; metabolism ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; genetics ; metabolism ; Tablets ; administration & dosage ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; Wound Healing ; drug effects