Objective To construct recombinant plasmid pEGFP C1 antisense survivin and than transfect it to MKN 45 gastric cancer cells for studying the expression of survivin mRNA and its effects on apoptosis of cancer cells. Methods Recombinant pEGFP C1 antisense survivin plasmid was cloned and transfected to MKN 45 gastric cancer cells by lipofectamine. Apoptosis of gastric cancer cells was assayed by flow cytometry. The levels of survivin mRNA before and after transfection were determined by reverse transcription polymerase chain reaction analysis. Results After recombinant pEGFP C1 antisense survivin plasmid was transfected, the apoptosis of MKN 45 cells was increase, the cell number in G 2/M phase was decreased, and the level of survivin mRNA was inhibited. Conclusions pEGFP C1 antisense suvivin can promote the apoptosis of MKN 45 gastric cancer cells, that might attribute to the inhibition on cell proliferation and expression of survivin mRNA.

ObjectiveTo investigate the effects of comprehensive geriatric assessment (CGA) intervention on depression and anxiety in patients with benign prostatic hyperplasia (BPH).Methods90 patients with BPH (aged 65 years and over) from June 2009 to June 2010 were randomly assigned to control(n= 45) and CGA (n = 45) group.Control group received conventional therapy,while CGA group was given CGA and consultation therapy as well as conventional therapy.The scores of international were assayed and compared between two groups before and 6 months after treatment.ResultsThe scores of international prostate symptom score (IPSS),quality of life scale (QOLS),self-rating depression scale (SDS) and self-rating anxiety scale (SAS) in control (t= 11.0,5.84,7.08and 9.68,respectively) and CGA (t= 14.0,10.4,9.16 and 6.1,respectively)group 6 months after treatment reduced as compared with those before treatmen (all P ＜ 0.01).After 6 months of treatment,the scores of IPSS,QOLS,SDS and SAS in CGA group were lower than in control group (t= 4.25,5.55,3.45 and 2.88,respectively,all P＜0.01).ConclusionsCGA intervention can improve the quality of life in BPH patients and alleviate their depression and anxiety.

ObjectiveTo evaluate the diagnosis and treatment of postoperative pulmonary embolism(PE). MethodsWe retrospectively analyzed clinical manifestations,diagnosis,treatment and prognosis of 51 postoperative PE. Results36 PE (70.59％ ) developed after orthopaedic surgery or with malignant tumors within 1 week after surgery.Dyspnea or chest distress was the most common symptoms.Sudden death was common in patients with PE.Problems in diagnosis of PE included:poor assessment of deep vein thrombosis(DVT) before operation,and the value of beside echocardiography in the diagnosis of patients with suspected high-risk PE was not fully recognized. Twenty-three PE cases received only anticogulant treatment.Intravenous thrombolysis or percutaneous interventional techniques were undertaken in 3 each cases.Cardiopulmonary resuscitation(CPR) simply after sudden death due to postoperative PE was often unsuccessful.ConclusionsPostoperative PE is a common cause of death,currently available measures are often ineffective.The key lies in prevention especially in those of high-risk PE or suspected non-high-risk PE.

Objective: To improve the traditional preparation procedure of effervescent tablets in order to raise stabilization. Methods: The orthogonal design was used for improvement of process. Results: The optimum procedure condition was A 3B 2C 1. That is adding 7.5% citric acid, 11.25% sodium bicarbonate encapsulated by 5% PEG. Conclusion: The new procedure is superior to the traditional procedure, and it is suitable for the requirement of production on a large scale.

Objective To investigate the therapeutic effects and safety of beraprost sodium on vascular endothelial dysfunction in elderly patients with lower extremity arteriosclerotic occlusive disease (LEAOD).Methods A total of 80 patients with LEAOD were randomly divided into 2 groups:the treatment group and the control group (n=40 each group).All patients were treated with conventional antihypertensive,antidiabetic,hypolipidemic,antiplatelet drug therapy and non-drug therapy such as smoking cessation,foot care.In addition,patients in the treatment group received 40 μg oral beraprost sodium at a dose of 120 μg/day,3 times/day for 30 days.The changes in symptoms,vital signs,the function of liver and kidney,hemorheology,and serum nitric oxide (NO)were observed before and after the treatment.Results No adverse reactions happened among all patients.After 30 days treatment,there was a significant difference in the total efficacy between the treatment and control groups (80％ vs.35％,x2 =19.60,P＜0.01).The levels of plasma viscosity,erythrocyte aggregation index and serum NO in the control group were (1.70±0.19) mpas.s,5.69±1.08,(43.86±5.20) μmol/L,respectively before the treatment and (1.36±0.14) mpas.s,3.23±0.67,(56.84 ± 7.05) μmol/L,respectively after the treatment.The levels of plasma viscosity,erythrocyte aggregation index and serum NO in the treatment group were (1.71 ±0.18) mpas.s,5.70±1.04,(44.24±5.40) μmol/L,respectively before the treatment and (1.10±0.12) mpas.s,2.80 ±0.52,(64.00±8.15) μmol/L,respectively after the treatment.Compared with pretreatment,the levels of plasma viscosity and erythrocyte aggregation index in the two groups were significantly decreased and serum NO level was increased after the treatment (t=9.07,12.17,17.85,15.76,9.38,12.78,respectively,all P＜0.05).Compared with the control group,the levels of plasma viscosity and erythrocyte aggregation index in the treatment group were significantly decreased and serum NO level was increased (t =8.91,3.27,4.21,all P＜0.05).Conclusions The oral beraprost sodium can effectively and safely improve the hemorheologic and endothelial function in elderly patients with LEAOD.

Aim To establish a LC-MS/MS method for the determination of hydroxysafflor yellow A ( QA ) in human plasma. Methods After being added into 0. 2M ammonium acetate (1∶1,V/V), QA was extrac-ted using solid-phase extraction technique, and the eluent was directly injected into LC-MS/MS systems. Agilent ZORBAX SB C18 (3. 0 × 100 mm, 3. 5 μm) column and isocratic elution system composing of meth-anol and 0. 2 mM ammonium acetate (70 ∶ 30, V/V) provided chromatographic separation of QA and internal standard isorhamnetin-3-O-neohespeidoside ( SLS) fol-lowed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 611 . 131→490. 900 for QA and m/z 623. 032→298. 800 for SLS. Results The retention time of QA and SLS was 2. 7 min and 3. 9 min respectively, with no interference in human blank plasma. The proposed method showed good linearity over the concentration range of 8. 57 ~4185 μg·L-1 for QA with a correlation coefficient≥ 0 . 9949 . The lower limit of quantitation was 8. 570 μg ·L-1 . The intra-batch and inter-batch precision and accuracy were within 7%. The average matrix effect ranged from 115. 72% to 119. 06% with RSD less than 5%. The average extraction recovery ranged from 77. 75% to 80. 76% with RSD less than 5%. Stability of human samples after 4 h at room temperature, after the three freeze-thaw cycles and after 31 days at -70℃, and post-preparative stability of the processed sam-ples after 24 h was acceptable. Plasma samples with the concentration beyond the upper quantitation limit could be accurately determined after being diluted using 6. 25 times ( V/V ) of human blank plasma. Conclusion Our current LC-MS/MS method is sensitive, accurate and convenient, and is proved to be suitable for the sys-tematic study on clinical pharmacokinetics of QA.

Objective:To assess incidence of malnutrition and malnutrition risk of six department patients.Methods:The information of 1 200 patients were collected,200 in each of 6 departments in our hospital.Nutrition status was assessed according to Nutrition Risk Screening(NRS)published by ESPEN in 2001.Results:The incidence of malnutrition and malnutrition risk varied from 7.5% to 59% and 36% to 72% respectively in different department.Conclusion:The incidence of malnutrition is closely related to the kind and severity of the disease.It is nessissary to assess the nutrition status of high risk patients in time.NRS can be used simply and fastly in most inhospital patients.

BACKGROUND: The activation of hypothalamus-pituitary-adrenal cortex axis may play key role in the increasing expression of hypothalamic corticotropin-re-leasing hormone (CRH) during stress reaction. However by what way to induce the CRH expression in hypothalamic neuron, and whether CRH can activate hypothalamic neurons are still not very clear.OBJECTIVE: To observe the changes of intracellular cyclic adenosine monophosphate (cAMP) and cytoplasmic Ca2+ concentration in the hypothalamic neurons cultured in vitro due to exogenous CRH stimulation.DESIGN: Comparative observation experiment.SETTING: Research Institute of Surgery, Daping Hospital, Third Military Medical University of Chinese PLA; Department of Neurosurgery , Tiantan Hospital, Capital University of Medical Sciences MATERIALS: This experiment was carried out in the Research Institute of Surgery, Daping Hospital, Third Military Medical University of Chinese PLA between December 1999 and March 2002. Hypothalamus was obtained from fetus rat at pregnancy of 17 days for the in vitro culture of hypothalamic neurons.METHODS: Hypothalamic neurons were co-cultured with exogenous CRH,with or without pretreatment with specific CRH 1 receptor antagonist -CP-154526. hypothalamic neurons were randomized into: ① CRH (10-12,10-10, 10-8, 10-6 mol/L) stimulation group. ② CP-154526(500 μmol/L)pretreatment aud CRH ( 10-12, 10-10, 10-8,10-6 mol/L) stimulation group. ③Hypothalamic neurons in corresponding normal control group were exposed to the isotonic saline stimulation. PTI fluorescence image system was used to determine and analyze the change of cytoplasmic free Ca2+ concentration in hypothalamic neurons due to exogenous CRH stimulation and RIA was used to detect the neuronal cAMP content.MAIN OUTCOME MEASURES: ①Cytoplasmic free Ca2+ concentration in hypothalamic neurons. ②cAMP content in hypothalamic neurons.RESULTS: The cytoplasmic free Ca2+ concentration and cAMP content were relatively lower in the hypothalamic neurons in normal control group,which obviously increased due to CRH stimulation [(240±22),(153±11)nmol/L; (3.26±0.19),(0.44±0.02) pmol/dish,P ＜ 0.01];CP-154526 could remarkably suppress the CRH (10-6 mol/L)induced increase in cytoplasmic free Ca2+ concentration and cAMP content in hypothalamic neurons [Ca2+ concentration: (240±22),(171±16)nmol/L; cAMP content:(3.26±0.19), (2.33±0.21) pmol/dish, P ＜ 0.01].CONCLUSION: CRH can directly act on hypothalamic neurons via type 1-receptor,thereby increase the cytoplasmic free Ca2+ concentration and cAMP content in hypothalamic neurons,playing the key role in the modulation of the synthesis and secretion of CRH during the activation of hypothalamic neurons.

BACKGROUND: Through what signal pathway does corticotropin release hormone (CRH) regulate hypothalamic neuronal neuroendocrine activity during acute stress?OBJECTIVE: To probe into the regulatory effects of CRH on CREB secretion in hypothalamic neurons.DESIGN: Repetitive measurement design.SETTING: At Field Surgery Research Institute of Daping Hospital, Third Military Medical University of Chinese PLA; Neurosurgery Department,Tiantan Hospital Affiliated to the Capital University of Medical Sciences.MATERIALS: This experiment was carried out in Daping Hospital of Third Military Medical University of Chinese PLA between December 1999and March 2002. Rat fetuses were selected from Wister rats of 17-day gestation.METHODS: In vitro cultured cells were divided into the following groups:① CRH (10-12, 10-10, 10-8 and 10-6 mol/L) stimulation groups. ② Pretreated with nimodipine (5 μmol/L) or CP-154526 (500 μmol/L) followed by CRH (10-12, 10-10, 10-8 and 10-6 mol/L ) stimulation groups. ③ Corresponding control groups stimulated with isotonic physiological saline. PTI fluorescence imaging system was used to detect the changes of neuronal cytoplasmic free calcium concentration; meanwhile, Western blot technique was used to determine the changes of neuronal P-CREB content.MAIN OUTCOME MEASURES: ① Changes of neuronal cytoplasmic free calcium concentration. ② Changes of neuronal P-CREB content.RESULTS: The content of cytoplasmic free calcium in hypothalamic neurons was lower in normal control group, and it increased immediately after exogenous CRH stimulation. However, such increase could be suppressed by pretreatment with nimodipine or CP-154526 before CRH stimulation,and the increase of neuronal P-CREB content was also obviously suppressed.CONCLUSION: During acute stress, the combination of CRH with hypothalamic neuronal CRH 1 receptor leads to the opening of membrane Ltype calcium ions channels, thus enhancing the influx of calcium ions and increasing cytoplasmic free calcium ions content, which would further activate P-CREB signal transduction pathway in neurons. It suggests that CRH may play a vital role in hypothalamic neuronal activation.

Objective To monitor the autophagy in osteosarcoma cells by constructing three rLC3B fusion expression vectors,respectively.Methods Rat LC3B gene sequence was amplified by PCR and cloned into pEGFP-C 1 and pmCherry-C1 to construct the fusion expression vector of pEGFP-rLC3B and pmCherry-rLC3B.Subsequently,the EGFP-rLC3B sequence was obtained by PCR with the pEGFP-rLC3B as a template,and cloned into pmCherry-C 1,so the pmCherry-EGFP-rLC3B fusion expression vector was constructed.Three plasmids were transfected into U-2OS cells,and the starvation or Rapamycin was adopted to induce autophagy or the chloroquine or Baf-A1 was used to inhibit autophagy,to verify the above plasmids' function in autophagy detection by laser scanning confocal microscopy.Western blot was used to detect the endogenous LC3B and exogenous EGFPrLC3B,pmCherry-rLC3B and mCherry-EGFP-rLC3B,and to verify the correct expression of exogenous rLC3B and their function of autophagy detection.Finally,cleaved free EGFP was detected by western blot to evaluate the level of autophagic degradation.Results Three fusion expression vectors were constructed successfully through sequencing and restriction enzyme digestion validation.The starvation or Rapamycin was adopted to induce autophagy or the chloroquine or Baf-A 1 was used to inhibit autophagy in transfected U-2OS cells.Clear autophagosomes and autolysosomes were observed by laser scanning confocal microscopy.Endogenous LC3B and exogenous EGFP-rLC3B,pmCherry-rLC3B and mCherry-EGFP-rLC3B were detected through western blot.Finally,western blot verified that the expression of cleaved free EGFP was significantly up-regulated with the increase of starvation time.12 h group increased 1.05 times than the control group and 24 h group increased 1.56 times,showing that the levels of autophagic degradation increased.Conclusion EGFP-rLC3B can be used to detect autophagosome and evaluate the level of autophagic degradation.mCherry-rLC3B can be used to detect autophagosome and autolysosome,but can't distinguish autophagosome from autolysosome.The pmCherry-EGFP-rLC3B has an advantage in the detection of autophagic flux which can distinguish autophagosome from autolysosome.