ObjectiveTo establish an effective method for F9 gene mutation detection by using high resolution melting ( HRM ) curve analysis.Methods Peripheral blood samples of 55 hemophilia B (HB) patients were collected from Shanghai Ruijin Hospital during January 2005 to June 2010.Genomic DNA was extracted from the peripheral blood.PCR amplification combined with sequencing was used to identify the F9 gene mutations in 40 patients.HRM assay was established on the 21 DNA samples with known mutations in exonl to exon7 of F9 gene.Mutation scanning of exonl to exon7 by HRM combined with direct sequencing of exon8 was used in the molecular diagnosis of 15 HB patients with unknown F9 gene mutations.ResultsF9 gene mutation was detected in each of the 40 HB patients by direct sequencing.By HRM,the different melting curve patterns were identified in 19 out of 21 cases.The detection rate was about 90％.Through mutation scanning of exonl to exon7 by HRM combined with direct sequencing of exon8,F9 gene mutations were detected in all the 15 HB patients with unknown F9 gene mutations.Thirty-four F9 gene mutations had been identified in the 55 HB patients.ConclusionsA new strategy of HB genetic diagnosis,scanning mutations of exonl to exon7 combined with DNA sequencing of exon8 of F9 gene,is established in this study.The new strategy is efficient and reliable.

Objective To determine the role of activated status of peroxisome proliferator-activated receptorγ/nuclear factor-κB ( PPAR-γ/NF-κB ) in coagulation disorders induced by sepsis. Methods Forty male Sprague-Dawley ( SD ) rats were randomly divided into four groups, n = 10 in each group: control group, lipopolysaccharide ( LPS ) challenged group, rosiglitazone ( ROSI, selective agonist of PPAR-γ) pretreatment group, and GW9662 ( PPAR-γ antagonist ) pretreatment group. The sepsis model was reproduced by injection of 6 mg/kg LPS via sublingual vein, and the rats in control group were injected with 2 mL/kg normal saline. The rats in ROSI pretreatment group were given 0.3 mg/kg ROSI by sublingual venous injection followed by injection of LPS 30 minutes later;and in GW9662 pretreatment group rats were given 0.3 mg/kg GW9662 by sublingual venous injection followed by 0.3 mg/kg ROSI 15 minutes later, followed by injection of LPS 30 minutes later. Blood was collected at 4 hours after LPS administration, and the expressions of PPAR-γ and NF-κBp65 in peripheral blood mononuclear cell ( PBMC ) were determined with immunocytocheminal technique and graph analysis. Plasma prothrombin time ( PT ), activated partial thromboplastin time ( APTT ), fibrinogen ( FIB ), and D-dimer were determined simultaneously. Results① PPAR-γ/NF-κB pathway: the expressions of PPAR-γ and NF-κBp65 were lowered in control group, and they were expressed in cytoplasm. In LPS challenged group the expression of PPAR-γ ( gray value ) was slightly increased but with no significant difference as compared with control group ( 111.01±4.06 vs. 98.46±5.99, P >0.05 ). In ROSI pretreatment group the expression of PPAR-γ( gray value ) was significantly higher than that in LPS challenged group ( 214.38±5.79 vs. 111.01±4.06, P<0.01 ), with dislocation into nuclei. In GW9662 pretreatment group the expression of PPAR-γ ( gray value ) was lowered but without significant difference compared with that of control group ( 44.21±2.64 vs. 98.46±5.99, P>0.05 ). In LPS challenged group the expression of NF-κBp65 ( gray value ) was significantly higher than that in control group ( 249.48±6.86 vs. 105.81±10.19, P < 0.01 ), and it was translocated into the nuclei. In ROSI pretreatment group the expression of NF-κBp65 ( gray value ) was significantly lower than that in LPS challenged group ( 102.47±8.05 vs. 249.48±6.86, P < 0.01 ), and it lied in cytoplasm. In GW9662 pretreatment group the expression of NF-κBp65 ( gray value ) showed no significant difference as compared with that of LPS challenged group ( 214.84±7.91 vs. 249.48±6.86, P>0.05 ).②Coagulation:compared with control group, PT and APTT were significantly prolonged, FIB was significantly decreased, and D-dimer was significantly increased in LPS challenged group [ PT ( s ):18.32±2.03 vs. 12.22±1.38, APTT ( s ):40.05±2.72 vs. 26.64±2.73, FIB ( g/L ): 1.65±0.51 vs. 3.60±0.37, D-dimer ( mg/L ): 2.58±0.73 vs. 0.37±0.06, all P < 0.01 ]. Compared with LPS challenged group, APTT and PT were significantly shortened, FIB was significantly increased, and D-dimer was significantly lowered in ROSI pretreatment group [ PT ( s ):13.93±1.67 vs. 18.32±2.03, APTT ( s ):30.29±0.86 vs. 40.05±2.72, FIB ( g/L ):3.18±0.69 vs 1.65±0.51, D-dimer ( mg/L ):0.40±0.12 vs. 2.58±0.73, all P<0.01 ]. All parameters in GW9662 pretreatment group showed no significant difference as compared with those of LPS challenged group. Conclusions PPAR-γagonist ROSI may ameliorate coagulation disorders in septic rats. PPAR-γ/NF-κB transduction pathway plays an important role in septic coagulopathy.

Objective To compare the immunostaining of human epidermal growth factor receptor-2(HER-2) among three different clones,and to compare with the results of fluorescence in situ hybridization(FISH) detection,to provide evidences for pathological department to select appropriate antibodies.Methods 268 cases of invasive breast cancer were examined by immunohistochemistry using antibodies for HER-2 of 3 different clones.The results were compared with those done by FISH.Results Immunostaining using antibody clone SP3 showed 3+ reactions in 66 cases;Immunostaining using antibody clone EP3 showed 3+ reactions in 53 cases,while using polyclonal HER-2 antibody,80 cases showed 3+ reactions.There was statistical significance between those groups using different clones(P<0.05).Consistent with the FISH results,the Kappa values are 0.76,0.67,and 0.56,respectively.Conclusion It suggests detection of HER-2 using immunohistochemistry should prioritize selecting monoclonal antibodies,especially clone SP3.

Objective: To construct HBV adr gene vaccine pCMVS 2-S and to observe the specific humoral immune responses in C57BL/6 mice after gene immunization. Methods: The HBV gene vaccine was injected into tibialis anterior muscles of C57BL/6 mice. ELISA was used to detected the anti-HBs antibody in mice sera at various time points after gene transfer. Results: Anti-HBs in sera of mice was tested positive 1 week after plasmid injected, the level of antibody peaked 4 weeks later, and kept high titer for at least 2 months. Conclusion: The good response of humoral immunity can be induced by injection of pCMVS 2-S in C57BL/6 mice.

A review of studies on the influence of impurities on protein crystallization is given.The possible sources of impurities and its effect on the protein crystallization are presented.The effects of impurities on protein crystallization,including nucleation,macroscopic morphologies,microscopic surface morphologies,growth rates,kinetics,quality,and repartitioning of impurities are reviewed.

The 3T3-L1 preadipocytes were used as carriers in the investigation of total extract, n-butanol extract, CB-1 and CB-2 of Coreopsis tinctoria Nutt. on cell proliferation and differentiation. Three groups at different doses were set for each of the four extract regions of C. tinctoria Nutt., respectively. MTT assay was used to detect 3T3-L1cell proliferation by four extract regions of C. tinctoria Nutt. Oil Red O staining was used to analyze the formation and accumulation of cytoplasmic lipid during cell differentiation. The results showed that compared with the control group, there were significant inhibition on cell proliferation when thetotal extract of C. tinctoriaNutt. at 100 μg·mL-1, n-butanol extract at 0.5, 5, and 50 μg·mL-1, CB-1 and CB-2 at 50 μg·mL-1 (P< 0.01). N-butanol extract showed certain dose-dependent manner (r = -0.903). Oil Red O staining showed that compared with the control group, thetotal extract of C. tinctoria Nutt. at 1, 10, 100 μg·mL-1 can obviously inhibit cell differentiation, reduce the formation of cytoplasmic lipid (P< 0.01). N-butanol extract can inhibit cell differentiation in a dose-dependent manner (r= -0.779). CB-1 and CB-2 obviously inhibited cell differentiation at the concentration of 50 μg·mL-1 (P < 0.01). It was concluded that thetotal extract, n-butanol extract, CB-1 and CB-2 of C. tinctoria Nutt. can inhibit the proliferation and differentiation of 3T3-L1 preadipocytes and reduce the formation of cytoplasmic lipid.

OBJECTIVETo investigate the clinical outcomes of the posterior C1,2 screw-rod combined with C2 unilateral translaminar screw and contralateral pedicle screw fixation and autogenous bicortical iliac crest graft fusion in treating upper cervical instability with vertebral artery variations.

METHODSFrom June 2008 to December 2012, the clinical data of 12 patients with upper cervical instability underwent C1 lateral mass screws-C2 unilateral laminar and contralateral pedicle screws fixation combined with autogenous bicortical iliac crest graft fusion were analyzed retrospectively. There were 8 males and 4 females with a mean age of 47.5 years (ranged, 16 to 77 years). Patients suffered from occipitocervical activity limitation of motion with pain or not, VAS was 0-7 points with an average of (3.50 +/- 2.71) points. Unilateral vertebral artery hypoplasia was demonstrated by vertebral arteriography (VAG) or CTA in all patients. Cervical X-ray and CT scans were done within 7 days after surgery in order to confirm internal fixation position. Internal fixation loosening and breakage, reduction losing, bone fusion ratio were observed during follow-up.

RESULTSNo nerves and vertebral artery injuries occurred during operation. Cervical pain obviously decreased and VAS was (0.92 +/- 0.90) points. Cervical alignment of 12 patients had well-recovered by X-ray while Atlantoaxial ventral lamina cortex of 1 case was encroached by CT scan without neurological symptom. All patients were followed up for 6 months to 3 years, no internal fixation loosening and breakage, reduction losing were found. All patients obtained bone fusion in 6-12 months after operation.

CONCLUSIONPosterior C1 lateral mass screws-C2 unilateral laminar and contralateral pedicle screws fixation combined with autogenous bicortical iliac crest graft fusion can achieve biomechanical stability and raise the successful rate of bone fusion, while avoiding the risk of vertebral artery injury and overcoming the insufficient of bone fusion during bilateral laminar screws placement as well. Posterior C1 lateral mass screws fixation is a safe and effective additional method in treating upper cervical instability with vertebral artery variations.

Adolescent ; Adult ; Aged ; Bone Screws ; Cervical Vertebrae ; surgery ; Female ; Humans ; Internal Fixators ; Joint Instability ; surgery ; Male ; Middle Aged ; Tomography, X-Ray Computed ; Vertebral Artery ; pathology

OBJECTIVETo study the effect of electro-acupuncture (EA) at Neiguan (PC6) and Lieque (LU7) on the expression of protein kinases in cardiomyocytes of myocardial ischemia (MI) rats.

METHODSHealthy male SD rats were randomly divided into the control group, the model group, the Neiguan point group, the Lieque point group, and the non-meridian non-acupoint group, 10 in each group by random digit table. The MI rat model was established by injecting isoprenaline hydrochloride (85 mg/kg). EA at Neiguan (PC6), Lieque (LU7), and non-meridian non-acupoint were respectively performed. Changes of the expression of protein kinases [such as protein kinase A (PKA), protein kinase C (PKC), protein kinase G (PKG)] in rat cardiomyocytes were observed using Western blot.

RESULTSCompared with the control group, expression levels of PKA, PKC, and PKG increased obviously in the model group (P < 0.01). Compared with the model group, expression levels of PKA, PKC, and PKG decreased in the Neiguan point group and the Lieque point group (P < 0.01, P < 0.05). Expression levels of PKA decreased in the non-meridian non-acupoint group (P < 0.01). Compared with the Neiguan point group, expression levels of PKA, PKC, and PKG increased in the non-meridian non-acupoint group and the Lieque point group (P < 0.01, P < 0.05). Compared with the Lieque point group, expression levels of PKA, PKC, and PKG increased in the non-meridian non-acupoint group (P < 0.01, P < 0.05).

CONCLUSIONEA at Neiguan (PC6) and Lieque (LU7) could decrease protein expression levels of PKA, PKC and PKG in rat myocardial cells, and the effect of acupuncture at Neiguan (PC6) was better than that obtained by EA at Lieque (LU7).

Acupuncture Points ; Acupuncture Therapy ; Animals ; Coronary Artery Disease ; Cyclic AMP-Dependent Protein Kinases ; Electroacupuncture ; Male ; Meridians ; Myocardial Ischemia ; metabolism ; therapy ; Myocytes, Cardiac ; metabolism ; Plant Extracts ; Protein Kinases ; metabolism ; Rats ; Rats, Sprague-Dawley

High price and poor stability of both crocin-1 and crocin-2 reference substance have become obstacles to HPLC assay of Croci Stigma. A new method based on reference extract was proposed. In this study, the reference extract was prepared from gardenia yellow which is cheap and easy to get The content of crocin-1 and crocin-2 in reference extract was determined and factors affecting stability of reference extract were investigated. Twelve batches of Croci Stigma were analyzed with reference extract and reference substance respectively. The results showed no difference. The presented method is feasible for quality control of Croci Stigma and reference extract is suitable to replace reference substances in assay.
Carotenoids ; analysis ; Chromatography, High Pressure Liquid ; standards ; Crocus ; chemistry ; Drugs, Chinese Herbal ; analysis ; Quality Control ; Reference Standards

Objective To investigate the therapeutic effects and toxic effects of radiochemotherapy versus chemotherapy alone in patients with advanced gastric cancer after gastrectomy.Methods A total of 183 patients with stage Ⅱ-Ⅳ (M0) gastric cancer,who received treatment after gastrectomy from 2004 to 2010,were included in the study.Of the 183 patients,64 received radiochemotherapy after gastrectomy,and 119 received chemotherapy alone after gastrectomy.The survival rates and hematological and gastrointestinal toxic effects were compared between the two groups;survival difference was also analyzed after the patients were stratified by TNM stage,number of metastatic lymph nodes,and extent of lymph node dissection (D0,D1,and D2).The Kaplan-Meier method was used for calculating survival rates,and the log-rank test was used for survival difference analysis and univariate prognostic analysis;the chi-square test was used for comparing toxic effects between the two groups.Results The follow-up rate was 87.8％ ;72 patients were followed up for at least 3 years,and 29 patients for at least 5 years.The 1-,3-,and 5-year survival rates for the radiochemotherapy group were 86％,62％,and 55％,respectively,as compared with 77％,53％,and 43％ for the chemotherapy group (P =0.079).There were no significant differences in grade 3-4 hematological and gastrointestinal toxic effects between the two groups (P =0.363 and 0.617).The univariate analysis showed that radiochemotherapy had a significantly better survival benefit than chemotherapy alone in patients with stage ⅢB-Ⅳ (M0) gastric cancer,patients who underwent D0 lymph node dissection,and patients with more than 6 metastatic lymph nodes (P =0.022,0.025,and 0.021).Conclusions Compared with chemotherapy alone,radiochemotherapy tends to improve survival in patients with gastric cancer after gastrectomy,and its toxic effects are tolerable.The patients with stage ⅢB-Ⅳ (M0) gastric cancer,patients who undergo D0 lymph node dissection,and patients with more than 6 metastatic lymph nodes can benefit from radiochemotherapy.