BACKGROUND:Understanding the actions and underlying mechanisms of proteins in cel s and organisms and studies on the lumbar disc herniation (LDH)-associated proteins contribute to further clarify the LDH pathogenesis. OBJECTIVE:To analyze the serum proteome changes in LDH patients using two-dimensional gel electrophoresis-tandem mass spectrometry, and to screen the biomarkers for LDH diagnosis and treatment. METHODS:Twenty-five LDH patients were enrol ed and their serums were col ected before and after treatment. After removal of high abundant protein, the serum protein spectrum was compared after two-dimensional gel electrophoresis serum protein spectrum diagram, to look for differential protein spots. Subsequently, the differential protein spots were identified by MALDI-TOF/TOF technique combined with biology software and database retrieval. RESULTS AND CONCLUSION:Six differential protein spots were screened preliminarily, and three kinds of proteins were confirmed after mass spectrum detection, among which, acid glycoprotein was related to the LDH immune regulation and occurrence, development and targeting sites of chemical radiculitis. The expression level of acid glycoprotein was significantly decreased in LDH patients after treatment (P<0.05). These results suggest that acid glycoprotein is associated with LDH through gel electrophoresis-base proteome analysis.

In order to evaluate the neuroprotective effect of Rosiglitazone Maleate (RSG) against brain ischemic injury, the effects of Rosiglitazone Maleate on the inflammation following cerebral ischemia/reperfusion were investigated. Focal cerebral ischemia was induced by the intraluminal thread for cerebral middle artery (MCA) occlusion. Rosiglitazone Maleate at concentrations of 0.5, 2 and 5 mg/kg was infused by intragastric gavage twice immediately and 2 h after MCA occlusion, respectively. The effects of Rosiglitazone Maleate on brain swelling, myeloperoxidase and interleukin-6 mRNA level in brain tissue after MCA occlusion and reperfusion were evaluated. The results showed that as compared with the model control group, RSG (0.5 mg/kg) had no significant influence on brain swelling (P>0.05), but 2 mg/kg and 5 mg/kg RSG could significantly alleviate brain swelling (P<0.05). All different doses of RSG could obviously reduce MPO activity in brain tissue after MCA occlusion and reperfusion in a dose-dependent manner. RSG (0.5 and 2 mg/kg) could decrease the expression levels of IL-6 mRNA in brain tissue after MCA occlusion and reperfusion to varying degrees (P<0.05) with the difference being significant between them. It was concluded that RSG could effectively ameliorate brain ischemic injury after 24 h MCA occlusion and inhibit the inflammatory response after ischemia-reperfusion in this model.

Abstract] Objective To establish an assay for the detection of porcine parvovirus ( PPV) and to verify its application for monitoring cells used for production.Methods A pair of primers and one probe were designed according to the conserved sequence encoding non-structural protein 1 (NS1).Based on the designed primers, a real-time fluorescent quantitative PCR assay for the detection of PPV was developed. Several parameters including the linearity, precision, minimum detection limit and anti-interference of the established assay were evaluated.A stock of PPV strains was prepared by infecting swine testicle ( ST) cells with PPV strains.An assay for the detection of PPV infection was developed by using ST cells as sensitive cells.A combined ST cell infection-PCR test was developed by combining the ST cell infection assay with the real-time fluorescent quantitative PCR assay.The sensitivity of ST cell infection-PCR test was analyzed.The cell samples used for production of biological products were detected by using the established assay.Results The real-time fluorescent quantitative PCR assay was specific for the detection of PPV without cross-reaction to other species of parvovirus virus, SV40 virus and other porcine viruses.The linear range of the assay was 1×109-1×104 copies/μl with a R2 value more than 0.98.The sensitivity of the real-time quantitative PCR assay was 1×104 copies/μl.Both of the intra-and inter-coefficient of variation (CV) were less than 5%in Ct values.The intra-and inter-CV in copies of detection were 5%-15% and 30%-40% respectively.The minimum detection limit of the real-time fluorescent quantitative PCR assay was 1CCID50/ml.The PPV strains were detected in cell samples with no interference.The sensitivity of ST cells infection-PCR test was 0.01CCID50/ml.All of the 22 cell samples were negative for PPV by using the real-time fluorescent quanti-tative PCR assay.Conclusion The real-time fluorescent quantitative PCR and the ST cell infection-PCR test for the detection of PPV in cells were established successfully.The application of the two assays was conducive to further enhance the safety of using cells for production and therapy.

Objective To establish an assay for the detection of Torque teno sus virus ( TTSuV) strains and to analyze its preliminary application to biologics.Methods Primers and probe were designed according to the conserved sequences.A fluorescent PCR assay for the detection of TTSuV strains was estab-lished.Several parameters including the specificity, linearity, accuracy, sensitivity and anti-interference of the established assay were verified.The fluorescent PCR assay was performed to detect the samples of por-cine blood, cell substrate and rotavirus vaccine.The porcine blood samples positive for TTSuV strains were further genotyped.Results The established fluorescent PCR assay was confirmed to have high specificity as no cross-reactions with parvovirus virus of various species, SV40 and porcine circovirus strains were detec-ted.The linear range of the assay was 1×109-1×103 copies/μl for TTSuV1 genotype and 1×109-1×102 cop-ies/μl for TTSuV2 genotype with a R2 value more than 0.993.The sensitivity of the fluorescent PCR assay was 1×103 copies/μl for TTSuV1 genotype and 1×102 copies/μl for TTSuV2 genotype.The intra-and inter-CVs were both less than 7%in Ct values and less than 25% and 45% respectively in copies.No interfer-ence was found in the detection of TTSuV nucleic acids from cell samples.8 out of 20 porcine blood samples were positive for TTSuV strains, among which one sample was positive for TTSuV1 genotype, four samples were positive for TTSuV2 genotype and the rest were positive for both TTSuV1 and TTSuV2 genotypes.Com-pared with the reference strain, strains genotyped as TTSuV1 and TTSuV2 were respectively shared 98%-99%and 98%homologies in sequences.All of the cell substrate and rotavirus vaccine samples detected by the fluorescent PCR assay were negative for TTSuV strains.Conclusion The fluorescent PCR assay for the detection of TTSuV was established successfully, the application of which would further improve the safety of biologics.

Objective To investigate the risk factors and treatment for infections after spinal internal fixation surgery. Methods The clinical data of 472 patients who underwent spinal internal fixation surgery from January 2012 to December 2012 was analyzed retrospectively, an average age of 50. 6 years (38~78 years). All cases were underwent posterior procedures. All infected patients received emergency opera-tion of wound debridement, drainage and sensitive antibiotic treatment. The mean follow-up time was 11 months (8~19 months). Risk fac-tors and treatment for infections were summarized and discussed. Results Of 472 patients,postoperative infections occurred in 9 cases with the infection rate of 1. 91%. The operation time,intraoperative blood and postoperative drainage was 100~325 min,200~1500 mL and 65~1350 mL,respectively,which were greater than the similar surgeries of same period. The initial signs of wound infection was observed at 10 d (6~16 d) after surgery. CRP,ESR and WBC were significantly increased in 4~7 d after surgery,and maintained at high level at least for 14 d. Bacterial culture results showed infection bacteria were mainly common skin flora. One infection recurred during followed-up and subse-quent treatment was successful. Conclusion Wound infection after internal fixation mainly occurred in the posterior procedure of spine, which were deep infection. The main clinical manifestation was the wound exudate and local deep tenderness,fever and wound surface swelling were relatively rare. Increased intraoperative bleeding,postoperative drainage volume,operation time were the risk factors,which lead to perio-perative malnutrition and subsequent infections. Debridement,drainage,and intravenously sensitive antibiotics could obtain an ideal outcome for most cases. It was not necessary to remove the internal fixation instrument and bone grafting.

Objective To compare the therapeutic effect of inhaled aerosolized and intravenous milrinone,a phosphodiesterase-3 inhibitor in rats with oleic acid-induced acute lung injury (ALI) .Methods Forty male SD rats weighing 300-350 g were randomly divided into 4 groups (n=10 each) : group Ⅰ normal control: group Ⅱ ALI; group Ⅲ milfinone inhalation and group Ⅳ intravenous milrinone.The animals were anesthetized with intraperitoneal 2% pentobarbital 40 mg/kg,tracheostomized and mechanically ventilated (FiO2 30%,VT 10 ml/kg,RR 80 bpm,I:E=1:2).The chest was opened and the heart was exposed.Pulmonary artery was catheterized via fight ventricle.MAP,CVP,airway pressure and pulmonary artery pressure (PAP) were monitored.ALI was induced with 10% oleic acid 2 ml/kg administered through fight external jugular vein in group Ⅱ,Ⅲ and Ⅳ.In control group 0.1% BSA solution 2 ml/kg was administered iv instead of oleic acid.In group Ⅲ at 30 min after oleic acid administration aerosolized milrinone 1 mg/ml was inhaled 4 times at 60 min interval.Each time milrinone was inhaled for 10 min.In group Ⅳ at 30 min after oleic acid administration a bolus of 10 μg/kg milrinone was given iv followed by 10 min milrinone infusion at 1 μ·kg-1·min-1.The same procedure was repeated 4 times at 60 min interval.MAP and PAP were recorded and blood samples were taken from carotid artery and pulmonary artery for blood gas analysis at the 1st,2nd,3rd and 4th treatment.PaO2/FiO2 and Qs/Qt were calculated.The animals were sacrificed by exsanguination after the 4th treatment.The lungs were removed.The left lung was lavaged.Neutrophil count and protein content in broncho-alveolar lavage fluid (BALF) were determined.W/D lung weight ratio and lung myeloperoxidase (MPO) activity were measured.The uhrastructure of the lung was examined with electron microscope.Results The MAP was significantly lower after oleic acid adminstration in group Ⅳ than in other 3 groups.PaO2/FiO2 was significantly decreased and Qs/Qt increased by iv oleic acid in group Ⅱ ,Ⅲ and Ⅳ.PAP was significantly increased after iv oleic acid in group Ⅱ ,Ⅲ and Ⅳ but was significantly lower in group Ⅲ and Ⅳ than in group Ⅱ .The neutrophil count and total protein content in BALF,W/D ratio and lung MPO activity were significantly increased in group Ⅱ ,Ⅲ and Ⅳ as compared with control group(Ⅰ) and were significantly higher in group Ⅳ than in group Ⅲ.The lung damage induced by oleic acid was less serious in group Ⅲ and Ⅳ than in group Ⅱ .Conclusion Inhaled aerosoLized milrinone has better therapeutic effect than intravenous milrinone in rats with oleic acid-induced ALI and is safer.

Objective To optimize and compare four in vitro cytotoxicity assays in order to find a relatively rapid assay that can replace the traditional 51Cr release assay to evaluate the cytotoxicity of immune cells prepared for immunotherapy.Methods Four assays including BATDA, CAM (calcein acetoxymethyl ester), CytoTox-Glo and PKH were optimized and used to measure the in vitro cytotoxicity of NK-92 cells to K562 cell line.Intra-and inter-assay reproducibility of these assays and their correlation with 51Cr release assay were analyzed.Results After optimization, all of the four cytotoxicity assays showed good correlation with effector to target (E/T) ratio in a certain range.Compared with the other three assays, CytoTox-Glo assay showed obvious hook effect at a high E/T ratio of 40∶1.BATDA assay could detect the significant cytotoxicity of NK-92 cells to K562 cells after incubating them for one hour and that was the shortest time taken by the four assays to detect in vitro cytotoxicity.Both CAM and PKH assays took about four hours and CytoTox-Glo assay took six to eight hours to detect the significant cytotoxic potency.All of the four assays, especially BATDA and CAM assays, showed good intra-and inter-assay reproducibility.Among these assays, BATDA assay showed the best correlation with the traditional 51Cr release assay.BATDA assay, as compared with the other three assays, could be used to detect the cytotoxicity of Caov3 cells, an adherent cell line, and showed good results in evaluating the cytotoxic potency of autologous primary NK cells and CD19-CAR T cells.Conclusion Compared with the other three assays, BATDA assay shows the best linear correlation with 51Cr release assay and has the advantages of time-saving and good reproducibility.Besides, it is a better assay for detecting the cytotoxicity of adherent cells.BATDA assay is a promising substitute for 51Cr release assay in evaluating the in vitro cytotoxic potency of NK cells and other immune cells.

Objective To estimate the excess relative risk coefficients of stomach cancer for Chinese population attributable to ionizing radiation.Methods The excess relative risk and excess absolute risk coefficients of stomach cancer were estimated based on Life Span Study by using risk models developed by BEIR Ⅶ committee (Biological Effect of Ionizing Radiation).Guided by transportation methods from Life Span Study to Americans,we determined that transportation method for Chinese population includes both multiplicative and additive models with a weight of 0.7 and 0.3 respectively,on an arithmetic scale.Besides,curve fitting was used to obtain sex-age-specific stomach cancer baseline incidence based on Chinese cancer annual report.Then,Chinese excess relative risk coefficients of stomach cancer were obtained by substituting excess relative risk,excess absolute risk of Life Span Study and Chinese baseline incidence rate into risk transportation model.Results Excess relative risk coefficients of stomach cancer for Chinese population are 0.26/Sv for male and 0.64/Sv for female,whose exposure age is 30 years old and cancer age is 60 years old.Coefficients increase with decreased exposure age and cancer age.Conclusions Excess relative risk coefficients of stomach cancer for Chinese population are by larger higher than that of Life Span Study,and their sex-age tendency are similar.

Humans ; Aconitine/analysis* ; Chromatography, High Pressure Liquid ; Poisoning/diagnosis*

Objective: To identify the functional domain of ?-Synuclein in affecting mitochondrial function and how the function to be impaired,especially,the mitochondrial membrane potential and the release of Cytochrome c.Methods: Harvest of ?-Syn-N and ?-Syn-△N by PCR,then subcloned into the pCMV-Myc mammalian expression vector.The recombinant plasmids were transfected into HEK293T cells by Lipofectamine 2000.After detecting the protein expression by Western blot,the functional domain was detected by co-immunoprecipitation.The mitochondrial membrane potential through flow cytometry and immunofluorescence,at the same time,the release of Cytochrome c through flow cytometry to detect.Results: The recombinant plasmids were constructed successfully.CO-IP has proved that N-terminal may be the functional domain of ?-Synuclein in affecting mitochondria.Over-expression of N-terminal could depolarize the mitochondrial membrane potential and induce the Cytochrome c releasing in MN9D cells.Conclusion: N-terminal may be the functional domain of ?-synuclein and over-expression of N-terminal could decrease mitochondrial activity.