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.

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.

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.

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.

Objective:To investigate MAML2 gene rearrangement, gene fusion patterns, and the clinicopathological characteristics of primary pulmonary mucoepidermoid carcinoma (PMEC).Methods:Forty-six cases of primary PMEC from Fudan University Zhongshan Hospital and Fudan University Shanghai Cancer Center between 2017 and 2020 were collected. MAML2 gene rearrangement in all cases was detected by fluorescence in situ hybridization (FISH). In 20 cases, MAML2 fusion patterns were detected by targeted RNA sequencing (RNAseq). The relationship between MAML2 gene rearrangement, fusion patterns, clinicopathological characteristics, and prognosis was analyzed.Results:The average age of PMEC patients was 41 years (range 15-71 years); the ratio of male to female was about 1.1 ∶ 1.0. Most PMECs were low grade in histopathology with an early clinical stage (stageⅠ-Ⅱ).The overall positive rate of MAML2 gene rearrangement detected by FISH was about 80.4% (37/46), and the rate was higher in low-grade PMEC (91.7%, 33/36). Of the 20 cases detected by RNAseq, all the 19 FISH positive cases showed gene fusion, mainly CRTC1-MAML2 fusion (16/19), the other three cases showed CRTC3-MAML2 fusion (3/19), the break point of all the fusion patterns was CRTC1/3 (exon 1)-MAML2 (exon 2); No gene fusion was detected in the single FISH negative case; Compared with the MAML2 FISH negative patients, the PMECs carrying CRTC1-MAML2 fusion were more commonly found in patients age ≤ 40 years, maximum tumor diameter ≤ 2 cm, low histopathological grade and early clinical stage (all P<0.05); The three PMECs carrying CRTC3-MAML2 fusion gene were all female with early clinical stage; Univariate analysis showed that MAML2 gene rearrangement/fusion, onset age ≤ 40 years old, smaller tumor size, low histopathological grade, early clinical stage, no metastasis at diagnosis and surgical treatment were significantly correlated with overall survival ( P<0.05), but Cox regression analysis suggested that none of the above indicators were the independent prognostic factors for the survival of PMEC. Conclusions:The high incidence of MAML2 gene rearrangement in PMEC suggests that it is an important molecular diagnostic marker of PMEC. RNAseq confirms that CRTC1/3-MAML2 is the main fusion pattern in PMEC, suggesting that MAML2 fusion transcription may be an important driving factor of PMEC. MAML2 rearrangement/fusion and related clinicopathological characteristics are associated with good prognosis.

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 observe the clinical manifestation of 155 patients with hepatolenticular degeneration (HLD) complicated with epilepsy and the therapeutic effect of integrative Chinese and Western medicine treatment on them.

METHODSClinical manifestation of patients and its relationship with abnormalities in cranial CT and/or MRI were observed. Patients were treated by combined treatment of copper repellent with sodium dimercaptosulfonate 20 mg/kg per day by intravenous dripping, and modified Gandou Decoction (GDD) by oral intake and antiepileptics as well, after treatment for 8-10 courses, the clinical effect, copper levels in urine and serum were compared between groups.

RESULTSIn the 155 HLD patients, 96 were complicated with petit mal and 59 with grand mal. In the CT and/or MRI conducted in 72 patients, all showed abnormal images, besides such frequently met images as bilateral symmetrical basal ganglia focal lesion in 65 case-episode (90.3%) and brain atrophy of various degrees in 61 case-episode (84.7%), the massive lesions in cerebral white matter as principal, with the cortex involved, were also found in 54 patients (74%), which were mostly bilateral and symmetric or located in 2 adjecent lobes of brain, the sites of damage, in sequence of occurrence, were frontal lobe, parietal lobe, temporal lobe and callosal gyrus. Brain atrophy was found in all the remained patients without above-mentioned lesions. Abnormal EEG was shown in 29 patients (40.2%), which mainly manifested as theta wave of moderate to high potential and/or short paroxysmal spike-slow or sharp-slow complex wave evoked. The urinary copper level in patients after treatment was 34.5 +/- 21.6 micromol/24 hrs, significantly higher than that before treatment, 4.49 +/- 1.93 micromol/24 hrs (P < 0.01). And the serum copper level in patients also lowered significantly (P< 0.01). Epileptic seizure was controlled completely along with the gradually improving of extrapyramidal symptoms.

CONCLUSIONPartial seizure was the most common type of seizure of HLD patient complicated with epilepsy, the next is systemic seizure. Cerebral damage lesion and obvious brain atrophy could be the main etiological factors of HLD complicated with epilepsy, combined copper repellent therapy of integrative Chinese and Western medicine, and antiepileptics produced good clinical effect on the patients.

Adolescent ; Adult ; Anticonvulsants ; therapeutic use ; Chelating Agents ; therapeutic use ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; therapeutic use ; Epilepsy ; complications ; drug therapy ; Female ; Hepatolenticular Degeneration ; complications ; drug therapy ; Humans ; Male ; Phytotherapy ; Unithiol ; therapeutic use