OBJECTIVE:To establish an HPLC method for the determination of the contents of Ligustrazine in rat's plasma,brain and liver.METHODS:Ligustrazine was separated on Hypersil ODS-C18 column with aspirin as internal standard.The mobile phase consisted of methanol-1.5% glacial acetic acid solution(45∶55,V/V)at a flow rate of 1.0 mL?min-1.The UV detection wavelength was 279 nm.RESULTS:The linear range of ligustrazine in rat's plasma,brain and liver was 0.006 25～7.813 ?g?mL-1,The lowest detectable limits were 0.5 ng?mL-1,1.55 ng?mL-1,and 1.55 ng?mL-1 and the average recoveries were 97.26%,96.44%,and 95.43% respectively with RSD at 3.40%,4.19% and 4.94%,respectively.CONCLUSION:With good linearity,precision and recovery,the method is sensitive and simple,and suitable for pharmacokinetic study and the research of Ligustrazine preparation.

Objective To analyze the relationship between age and the prognosis of patients with pancreatic neuroendocrine tumors (pNETs).Methods The clinical data of 102 patients with pNETs who were admitted to the Zhongshan Hospital of Fudan University from January 1999 to December 2010 were retrospectively analyzed.The properties of the tumors were determined by morphology and immunohistochemical staining of chromogranin A,synaptophysin and neuron-specific enolase.Preoperative grading of the tumors was done by mitotic count and Ki67 proliferation index,and the staging of the tumors was done by ENETS TNM system.All patients were divided into younger group (age≤60 years,77 patients) and older group (age ＞ 60 years,25 patients).The survival of the patients was estimated using the life table,and the survival curve was drawn by the Kaplan-Meier method.The survival of the 2 groups was compared using the Log-rank test.Multivariate analysis was performed with the COX proportional hazards model.Results The median survival time was 139.8 months.The overall 1-,2-,5-,and 10-year survival rates were 96％,93％,90％ and 80％,respectively.The postoperative survival time of patients in the younger group was significantly longer than that in the older group (x2 =4.717,P ＜ 0.05 ).The prognosis of patients with higher tumor grades ( G2,G3) and higher TNM stages ( Ⅲ,Ⅳ ) in the older group was significantly poorer than those in the younger group ( x2 =11.158,5.375,P ＜ 0.05 ).The results of multivariate analysis showed that age and major vascular invasion were the independent predictors for survival (RR =8.626,12.795,P ＜0.05 ).Conclusions Age above 60 years is an important independent factor influencing the prognosis of pNETs patients.Tumor grading and TNM staging are highly correlated with the prognosis of the pNETs patients.

Objective To investigate the clinicopathological features,treatment method and prognostic factors of the gastric neuroendocrine neoplasms (gNENs).Methods The clinical data of 80 patients with gNENs who were admitted to the Zhongshan Hospital of Fudan University from January 2002 to December 2011 were retrospectively analyzed.All the patients received gastroscopic examination.Patients with well-differentiated and diameter ≤ 2 cm gNENs received endoscopic mucosal resection or endoscopic submucosal dissection.Patients with poordifferentiated and diameter ＞ 2cm tumors received surgical resection of gNENs.Patients were followed up via phone call,mail or out-patient examination till October 31,2013.The Kaplan-Meier method was used for drawing the survival curve and calculating the survival rate.The univariate analysis was done by Log-rank test and the multivariate analysis was done by COX proportional hazards model.Results Fifty-eight patients had pain and discomfort in the epigastric region.Thirty-two gNENs were located at the cardia,40 at the body and 8 at the pylorus.Fortyfour gNENs were ulcerative type,25 were polypoid type,11 were protruded type.The mean diameter of the gNENs was 2.6 cm (range,0.4-7.5 cm).Twenty-seven gNENs were in grade 1,10 in grade 2 and 43 in grade 3.Forty-five gNENs were localized,34 gNENs had lymph node involvement (2 gNENs had distal metastasis),and 1 gNENs had distal metastasis.Thirty-one patients received endoscopic resection,and did not receive adjuvant therapy.Forty-nine patients (6 with gastric neuroendocrine tumor and 43 with gastric neuroendocrine carcinoma)received radical resection,including proximal subtotal gastrectomy in 16 patients,distal subtotal gastrectomy in 15 patients,total gastrectomy in 15 patients and distal subtotal gastrectomy + resection of the liver metastasis in 3 patients.Thirty-four patients received adjuvant chemotherapy and 15 did not receive adjuvant chemotherapy.Seventy-nine patients were followed up for a median time of 42.0 months (range,2.0-113.0 months).The mean time of survival was 75.6 months(range,2.8-100.8 months),and the 1-,3-,5-year overall survival rates were 91.3％,75.8％ and 66.5％,respectively.The 5-year survival rates of patients with gNENs in grade 1,grade 2 and grade 3 were 100.0％,100.0％ and 38.0％,respectively.The results of univariate analysis showed that the gender,treatment methods,adjuvant chemotherapy,types of tumor,tumor diameter,classification and staging of the tumor were correlated with the prognosis of the patients (x2=9.550,17.488,25.038,14.994,6.897,25.234,22.066,P ＜ 0.05).The results of multivariate analysis showed that the gender was the independent risk factor influencing the prognosis of the patients (RR =11.280,95％ confidence interval:5.353-19.121,P ＜ 0.05).Conclusions The clinical presentations of gNENs are often nonspecific.The main presentation of gNENs is pain in the epigastric region of the abdomen,and most of the gNENs are located at the cardia or body of the stomach.The staging and grading of the gNENs are varied,and the prognosis is related with the gender of the patients.Endoscopic or surigcal resection is the main treatment method for gNENs.Female patients have a better prognosis than male patients.

BACKGROUND:Recent studies have showed that S100A8 has been implicated in the pathobiology of inflammatory disorders, and that cerebral ischemia reperfusion (I/R) rapidly activates inflammation responses via Toll-like receptor 4 (TLR4). This study aimed to explore the expression of S100A8 and the relationship between S100A8 and TLR4 in focal cerebral ischemia reperfusion injury.METHODS:C3H/HeJ mice (n=30) and C3H/HeN mice (n=30) were divided randomly into a C3H/HeJ model group (n=18), a C3H/HeJ control group (n=12), a C3H/HeN model group (n=18), and a C3H/HeN control group (n=12). Middle cerebral artery I/R model in mice was produced using a thread embolism method. The brains of the mice were collected after ischemia for 1 hour and reperfusion for 12 hours. Stroke outcome was evaluated by determination of infarct volume and assessment of neurological impairment scores. Brain injury after cerebral I/R was observed by an optical microscope after TTC and HE dyeing. The immunofluorescence technique and real time PCR were used to test the expression level of S100A8 in brain damage.RESULTS:Compared with C3H/HeN mice, TLR4-deficient mice (C3H/HeJ) had lower infarct volumes and better outcomes in neurological tests. The levels of S100A8 increased sharply in the brains of mice after I/R injury. In addition, mice that lacked TLR4 (C3H/HeJ) had lower expression of I/R-induced S100A8 than C3H/HeN mice in the model group, indicating that a close relationship might exist between the levels of S100A8 and TLR4.CONCLUSION:S100A8 interaction with TLR4 might be involved in brain damage and in inflammation triggered by I/R injury.

BACKGROUND:This study was undertaken to observe the concentration of SP-A/B and the pulmonary surfactant in the lung tissue of rats with acute lung injury/acute respiratory distress syndrome caused by paraquat poisoning after the treatment of metabolic antioxidant-lipoic acid and whether its influence was related to TNF-α. METHODS:Sixty-six male Sprage-Dawley rats were randomly divided into three groups:normal control group (NS group), 6 rats; paraquat poisoning group (PQ group), 30 rats; and paraquat+lipoic acid treatment group (LA group), 30 rats. The rats in the PQ and LA groups were subdivided into 3-, 6-, 12-, 24-, 48-hour subgroups, with 6 rats in each group. After the rats were sacrificed, lung tissue from the same part was taken from the rats. After HE staining, histological changes were observed in the tissue under a light microscope. Lung tissue was also taken to test the levels of superoxide dismutase (SOD) and malondialdehyde (MDA). Whole blood (0.8 mL) without anticoagulant was drawn from the tail vein of rats for the determination of the TNF-α level. The total RNA of the lung tissue was collected, and the Rt-PCR method was used to measure the levels of SP-A and SP-B mRNA. RESULTS:HE staining showed that histopathological changes were milder in the LA group than in the PQ group. There were significant differences in MDA and SOD levels between different intervals both in intergroups and intragroups except the 3-hour subgroup (P<0.01). Likewise, the significant differences in the levels of TNF-α were also present between the three groups and between different intervals (P<0.01). The significant differences in SP-A mRNA and SP-B mRNA amplification ratio were seen between the three groups at the same intervals (P<0.01), but the differences between different intervals in the PQ group were statistically significant (P<0.05). The differences between different intervals in the LA group were statistically significant (P<0.01). CONCLUSION:Lipoic acid in acute paraquat poisoning could diminish lung tissue damage by regulating directly tumor necrosis factor and indirectly the content of pulmonary surfactant so as to reduce pulmonary edema, improve lung compliance, and finally protect lung tissues.

The Bcl-2 family of proteins play a central role in the control of apoptosis, a fundamental process for both human health and disease, by mitochondrial pathway. PUMA(p53 up-regulated modulator of apoptosis protein) is one of BH3-only members of Bcl-2 family , its function is to promote cell apoptosis. To obtain BH3 death domain peptide of PUMA and detect its biological activity, the synthesized double-stranded oligomeric nucleotide encoding PUMA-BH3 peptide was cloned into expression vector pTYB2,thus generating a construct of pTYB2-PUMA-BH3 which expressed PUMA-BH3-intein-chitin binding domain fusion protein. Then the recombinant plasmid was transformed into E.coli BL-21 (DE3) and fusion protein was expressed under induction by IPTG. The soluble PUMA-BH3 peptide was purified from chitin affinity chromatography by DTT reduction. Through measuring mitochondria viability(MTT),mitochondria permeability transition(MPT) and the translocation of cytochrome c(Cyt c ) assayed by western blotting, the biological pro-apoptotic activity of PUMA-BH3 peptide was studied. The PUMA-BH3 peptide has the effects on decreasing the mitochondria viability remarkably , inducing mitochondrial swelling and promoting Cyt c releasing from isolated mitochodria . Mitochondrial swelling and the release of Cyt c induced by PUMA-BH3 peptide concerned with the opening of MPT,which can be improved by cyclosporine A(CsA).These results indicated that recombinant PUMA-BH3 peptide might possess pro-apoptosis activity and paved a reasonable way for the study of new apoptosis regulators.

Objective To observe the histopathological and ultrastructural changes,alterations in the expressions of type Ⅳ collagenases(MMP-2 and MMP-9),the tissue inhibitors(TIMP-1 and TIMP-2)and intercellular adhesion molecule-1(ICAM-1)in the internal carotid arteries(ICAs)of patients with moyamoya disease(MD),and explore the pathogenesis of MD.Methods The bilateral ICAs were obtained during autopsy from two MD patients.HE staining and Weigert staining was used for histological observation,and transmission electron microscopy Was employed to observe the ultrastructure of the ICAS.The expression of ICAM-1 in the ICAs was detected using immunohistochemical staining,and the mRNA expressions of MMLP-2,MMP-9,TMP-1 and TIMP-2 were assayed using in situ hybridization.The ICA specimens from two patients died from non-vascular diseases were used as the control.Restilts HE staining revealed thinning of the ICAs of the MD patients with luminal stenosis,obstruction and calcification.Weigert staining identified fibrous thickening of the intima,thinning and fragmentation of the elastica interna(EI)and elastica externa(EE),degenerafion of the smooth muscle cells in the media,and thinning and local exfoliation of the adventitia.Immunohistochemistry showed ICAM-1 expression in the ICAs of both MD and control patients,but the MD patients exhibited a stronger ICAM-1 positivity in the ICAs localized primarily in the tunica intima.The ICA wall was positive for MMP-9 mRNA expression,which Was especially intense in the elastica intema,as shown by in situ hybfidization.Conslusions The high expression of MMP-9 and ICAM-1,destruction of the elastic layer and adventitia,and collapse of the vascular wall result in luminal stenosis or everi obstruction of the ICAs,which can be associated with the occuITence of MD.

The key points for better protection of trehalose in freeze-drying red blood cells (RBCs) are to resolve non-osmosis of trehalose to red blood cells and to make cytoplasmic trehalose to reach effective concentration. This study was aimed to investigate the regularity of loading RBCs with trehalose, screen out optimal loading condition and evaluate the effect of trehalose on physico-chemical parameters of RBCs during the period of loading. The cytoplasmic trehalose concentration in red blood cells, free hemoglobin and ATP level were determined at different incubation temperatures (4, 22 and 37 degrees C), different trehaolse concentrations (0, 200, 400, 600, 800 and 1000 mmol/L) and different incubation times (2, 4, 6, 8 and 10 hours), the cytoplasmic trehalose, free hemoglobin (FHb), hemoglobin (Hb) and mean corpuscular volume (MCV) in fresh RBCs and RBCs stored for 72 hours at 4 degrees C were compared, when loading condition was ensured. The results showed that with increase of incubation temperature, time and extracellular trehalose concentration, the loading of trehalose in RBCs also increased. Under the optimal loading condition, cytoplasmic trehalose concentration and free hemoglobin level of fresh RBCs and RBCs stored for 72 hours at 4 degrees C were 65.505 +/- 6.314 mmol/L, 66.2 +/- 5.002 mmol/L and 6.567 +/- 2.568 g/L, 16.168 +/- 3.922 g/L respectively. It is concluded that the most optimal condition of loading trehalose is that fresh RBCs incubate in 800 mmol/L trehalose solution for 8 hours at 37 degrees C. This condition can result in a efficient cytoplasmic trehalose concentration. The study provides an important basis for long-term preservation of RBCs.
Biological Transport, Active ; drug effects ; Blood Preservation ; methods ; Cryopreservation ; methods ; Cryoprotective Agents ; metabolism ; pharmacology ; Erythrocyte Membrane ; metabolism ; Erythrocytes ; Freeze Drying ; Humans ; Osmotic Fragility ; Temperature ; Time Factors ; Trehalose ; metabolism ; pharmacology

OBJECTIVETo study the biological effects of 585 nm pulsed dye laser (FLPDL) in the treatment of congestive scar.

METHODSBy histological study, collagen VG staining and microvascular staining, we investigated the changes of collagen fibers and the density of microvessels in the congestive scars after FLPDL treatment.

RESULTSHistological and immunohistochemistry examinations showed that FLPDL achieved normal vascularity in the scar after over 3 times of treatment.

CONCLUSIONSPDL treatment can change fundamentally the physiology of wound healing if applied in the early phases.

Adolescent ; Adult ; Cicatrix ; therapy ; Female ; Humans ; Lasers, Dye ; Low-Level Light Therapy ; methods ; Male ; Middle Aged ; Reconstructive Surgical Procedures ; methods ; Treatment Outcome ; Wound Healing ; Young Adult

OBJECTIVETo assess the effect of Bryan cervical disc replacement on the function of the cervical spine.

METHODSBryan cervical artificial disc replacement was performed in 164 cases from Dec 2003 to Aug 2007, and all the cases were retrospectively followed up. Among them, 1 disc replacement was done in 132 cases, 2 discs in 28 cases and 3 discs in 4 cases with a total number of 200 artificial discs. There were 102 male patients and 62 female patients. Their age ranged from 25 to 70 years old (with an average of 47 years old). All the cases were operated according to the standard procedure for Bryan artificial disc replacement, and immobilized in a cervical collar for 2 weeks after operation. Motion of the replaced disc in sagittal direction, JOA score and satisfaction rate of the patients were followed up and evaluated.

RESULTSIn this group, no acute complications happened during the operation. All patients returned to work 4 to 6 weeks after operation. The postoperative ameliorate rate of JOA score was 56%. Range of motion in sagittal direction of the operated disc was 14.4 degrees before operation, decreased to 5.7 degrees at 1 week after operation , but improved to 14.7 degrees at the time of final follow-up and was not significantly different from preoperative range. Motion in the upper adjacent disc to the replacement level was 10.9 degrees before operation, decreased to 5.5 degrees at 1 week after operation , and improved to 8.2 degrees at the time of final follow-up but was significantly smaller than preoperative range. The satisfaction rate of the patients was 94%. Loosening of the prosthesis happened in 1 case 6 months after operation but remained stable afterwards. Subsidence up to 1 mm occurred in another case 7 months after operation but also remained stable afterwards. Automatic posterior union occurred in 3 cases in which relative small size artificial discs were implanted.

CONCLUSIONSThe clinical outcome of Bryan artificial disc replacement was quite good. Pre-operative range of motion of the cervical spine can be maintained.

Adult ; Aged ; Arthroplasty, Replacement ; methods ; Cervical Vertebrae ; physiopathology ; surgery ; Female ; Follow-Up Studies ; Humans ; Intervertebral Disc ; surgery ; Joint Prosthesis ; Male ; Middle Aged ; Range of Motion, Articular ; Retrospective Studies ; Spinal Osteophytosis ; surgery