Objective To development a cooling crystallization process that is suitable for industrial preparation of purified dihydromyricetin.Methods Screen design was used to investigate effects of process parameters such as,temperature,concentration ethanol aqueous,quantity of activated charcoal and adsorption time on yield and purity of dihydromyricetin.Purity was verified by high performance liquid chromatography and thin layer chromatography.The solubility of dihydromyricetin in water at viable temperature and ethanol proportion was also determined by UV spectrophotometry.The solid form was characterized by thermal analysis and powder X-ray diffractometry.Results When temperature was ＞ 85 ℃,ethanol concentration ＜ 10％,dosage of activated charcoal 0.1％-0.3％,and adsorption time 1-3 min,yield of dihydromyricetin was more than 70％,and the purity greater than 98％.The crystals,prepared by cooling crystallization from water and ethanol aqueous,had the same physical form and crystal habit.Conclusion Cooling crystallization from low concentration of ethanol aqueous gets higher yield and the process is more robust than crystallization from water.

This study aimed to investigate the effects of fusion proteins GnRH-GRP(G3G6)and HSP65-STEAP1(HST1)on dendritic cells(DC)and the sensitization of DCs to B16F10 melanoma. The fusion proteins G3G6 and HST1 were obtained using the previous engineering strains in our laboratory. Group by unsensitized DC(US-DC), the G3G6 fusion protein sensitized DC, the HST1 fusion protein sensitized DC(HST1-DC)and the combined sensitized DC(GH-DC), the mouse bone marrow-derived DCs were sensitized with fusion protein to obtain the fusion protein sensitized DC vaccines. B16F10 melanoma cells were transplanted into C57BL/6J male mice to construct a melanoma model(1×106 cells per mouse), and DC vaccine was injected for treatment. The antitumor efficacy of DC vaccine was explored by in vitro and in vivo experiments. Flow cytometry analysis showed that the fusion protein can effectively stimulate DC into differentiation and maturation; in the animal experiment, the inhibition rate of melanoma treated with G3G6-DC was 35. 75%, that of HST1-DC group and combination group were 34. 03% and 55. 74%. It was initially proved that both G3G6-DC and HST1-DC can effectively inhibit the growth of transplanted tumors of melanoma B16F10 cells in mice, and the combination therapy is superior to the single therapy.

Objective:To establish an HPLC-MS/MS method for the determination of vinblastine in rat plasma. Methods:Aceto-nitrile was used to precipitate protein in the samples after the addition of internal standard, and then the concentration was analyzed by HPLC/MS/MS. All the separations were carried out on an Ultimate C18 column (150 mm × 2. 1 mm, 5. 0 μm). The mobile phase was composed of acetonitrile and 10 mmol·L-1 ammoniumacetate (containing 0. 1% formic acid) (49 ∶51) and was pumped at a flow rate of 0. 3 ml·min-1 under 40 °C. The detection was performed with multiple reactions monitoring (MRM) using electrospray ioniza-tion (ESI). The precursor/product ion transitions were monitored at m/z 811. 4→m/z 224. 2 (positive ion mode) for vinblastine and m/z 825. 4→m/z 807. 4(positive ion mode) for internal standard vincristine. Results:Good linearity of vinblastine was obtained with-in the range of 0. 457-950 ng·ml-1(r=0. 997 1). The lower limit of quantification was 0. 457 ng·ml-1. The extraction recoveries were within the range of 89. 15%-95. 28%. The precision of intra-and inter-day was not more than 7. 95%. T1/2 of vinblastine in rats was (5. 86 ± 2. 37) h, and AUC(0-t) and AUC(0-∞) was (68. 45 ± 14. 51) and (95. 03 ± 33. 09)μg·L-1 ·h, respectively. Conclu-sion:The method is fast, sensitive and accurate, which provides research basis for the development of vinblastine and transporters re-search in medicine. The concentration of vinblastine in rats is low, and the half-life is long.

OBJECTIVE:To establish physiological pharmacokinetic (PBPK) model of cefdinir in healthy volunteers,and to predict pharmacokinetic process of cefdinir in volunteers after oral administration. METHODS:Using“toubao dini”“cefdinir”“logP”“pKa”as keywords,related literatures about physico-chemical constants of cefdinir were retrieved from CNKI,ScienceDi-rect,PubMed and other databases;according to related guidelines and preliminary clinical trial plan of FDA,GastroPlusTM 8.6 soft-ware was used to establish PBPK model of oral administration of cefdinir;the effectiveness of the model was evaluated by multiple error. The model was used to simulate the absorption of cefdinir in the gastrointestinal tracts. The bioequivalence of test preparation and reference preparation were evaluated through single and population(n=500)simulation tests using cmax and AUC0-∞ of cefdinir reference preparation (capsule and granular formulation) as factors when release rate t85%=15 min (i.e. accumulatively released 85% within 15 min). RESULTS:The blood concentration-time curves of cefdinir predicted by PBPK model fitted well with mea-sured value(R2≥0.95);the pharmacokinetic parameters(cmax,tmax,AUC0-∞)were close to measured results,and the multiple er-rors were less than 2. After oral administration,cefdinir was mainly absorbed by the intestinal tract (45.6%),especially by seg-ment 1 of jejunum(14.8%);the absorption amount was significantly lower than the release amount of absorption site,and reached the maximal value(about 40%)within 4 h. The results of single simulation test showed that there was no statistical significance in cmax and AUC0-∞ between cefdinir test and reference preparations (P>0.05). The results of population simulation test showed that the relative bioavailability of cefdinir test particle and test capsule respectively were 99.01%-102.99% and 97.60%-105.90%;90%CI of cmax and AUC0-∞ values were within 80%-125% of reference preparation. CONCLUSIONS:The PBPK model is accurate and reliable in this study,can provide reference for pharmacokinetic study and bioequivalence evaluation of cefdinir preparations. Test preparation and reference preparation are equivalent.

Objective:To establish an LC–MS/MS method for the determination of the active metabolite(SN-38) and secondary metabolite(SN-38G) of irinotecan in rat liver microsomes incubation system, and optimize the incubation conditions. Methods:Meth-anol was selected to precipitate protein in the samples, and then the concentrations were analyzed by LC–MS/MS. All the separation was carried out on a ZORBAX Eclipse XDB-C18 column(2. 1 mm × 50 mm, 3. 5 μm) with the mobile phase of acetonitrile – water (containing 0. 1% formic acid) (23 :77) at a flow rate of 0. 3 ml·min-1. The mass spectrometer was operated with multiple reac-tions monitoring ( MRM) using electrospray ionization ( ESI) . The incubation conditions were optimized by single factor design. Re-sults:SN-38 and SN-38G showed a good linearity ( r≥0. 9972) respectively within the range of 2. 3-920 ng·ml-1 and 2. 5-1000 ng ·ml-1. The intra-and inter-day RSD was below 14. 6%(n=6). The average recovery was within the range of 74. 1%-123. 4% with RSD below 13. 5% (n=6). The optimal incubation conditions were as follows:the concentration of liver microsomal protein was 0. 3 mg·ml-1 and the incubation time was 30 min. Conclusion:The method is rapid, sensitive and accurate in the quantification of SN-38 and SN-38G in the incubation system,which provides methodological basis for the activity determination of UGT1A1 enzyme in vitro.

Objective:To evaluate the safety of cardiac catheterization intervention therapy and transthoracic small incision surgery in the occlusion bydomestic occluder under echocardiography guiding in patients with atrial septal defect (ASD).Methods:A total of 1 080 patients with ASD in the occlusion by domestic occluder were analyzed retrospectively,and the interventional treatment were performed in 734 cases through cardiac catheterization intervention therapy and 346 cases through transthoracic small incision surgery.The patients undergone cardiac catheterization intervention therapy were guided under the digital substraction angiography (DSA) and were monitored by transthoracic echocardiography (TTE) in the whole interventional process,and the efficacy was evaluated with TTE.The occlusion of transthoracic small incision surgery was guided under the transesophageal echocardiography (TEE),which was used to monitor the position of occluder and evaluate the efficacy immediately.Results:Two kinds of intervention in the occlusion by domestic occluder had achieved satisfactory results in patients with ASD.There was no statistically difference in the longest size of ASD between the 2 intervention methods,while there were statistically differences in the ratio between ASD longest diameter and atrial septal length,and the size of the occlusion,and the disparity between the size of the occluder and ASD longest diameter (D value),respectively (all P＜0.05).When the size of arithmetic mean of the ASD was ＜30 mm,the success rate of the 2 methods was both 100％.When the size of arithmetic mean of the ASD was ≥ 30 mm,the success rate was 100％ in the transthoracic small incision surgery and 50％ in the cardiac catheterization intervention therapy.Conclusion:Domestic occluder is safe.Compared with the imported one,its cost is lower.When the size of the defects is same,the occlusion is smaller in the transthoracic small incision surgery compared with that in the cardiac catheterization intervention therapy.When the size of arithmetic mean of the ASD is ≥ 30 mm,the success rate of the transthoracic small incision surgery is higher compared with the cardiac catheterization intervention therapy.When the cardiac catheterization intervention therapy fails,the transthoracic small incision surgery may be a better choice.

Objective To retrospectively analyze the clinical distribution and changes in antimicrobial resistance profiles of Staphylococcus aureus (S. aureus). Methods We collected clinical specimens of S. aureus from The First Hospital of China Medical University. The Vitek-2 and BD Phoenix 100 were performed for bacterial identification and drug sensitivity tests,and WHONET 5.6 was used to analyze the data. Results From 2007 to 2016,there were 3 377 unrepeatable strains of S. aureus,including 1 705 that were methicillin resistant S. aureus (MRSA). The isolation rate of S. aureus was 9.4 ％ and of these,50.5 ％ were MRSA. There were 776 S. aureus specimens from outpatients or the emergency department,including 16.8 ％ MRSA,and 2 011 S. aureus from inpatient departments,including 60.2 ％ MRSA. The main sources of specimens were sputum (41.8 ％),pus (17.9 ％),and body secretions (17.5 ％). The average resistance rates of MRSA for erythromycin,ofloxacin,ciprofloxacin,gentamycin,and tetracycline were higher than 75.0 ％. The average resistance rate of methicillin sensitive S. aureus (MSSA) for erythromycin was up to 76.8 ％,and for tetracycline,gentamycin,ciprofloxacin,and ofloxacin,were less than 25.0 ％. In 10 years,the average resistance rates of MRSA and MSSA for 11 kinds of common antibiotics had no obvious change. Conclusion The constituent rate of MRSA was high in The First Hospital of China Medical University,especially from the areas that were not sterile,suggesting that clinicians should pay attention to the identification of infection and sources for MRSA,which were from such areas. Hospital infection control should be focused on at the same time,in order to reduce the incidence of MRSA.

Objective ToevaluatethevalueofMRDKIandDWIindiagnosingendometrialcarcinomaandevaluatingitspathologicalgrade. Methods TheDKIandDWIdataof48patientswithendometrialcarcinomaand27patientswithnormalendometrium wereanalyzed retrospectively.Thevaluesofmeankurtosis (MK),meandiffusion (MD)andADCinendometrialcarcinomaandnormalendometrium were measuredrespectively.Thesimilaritiesanddifferencesoftheparametersbetweentheendometrialnormalgroup(G0)andtheendometrialcarcinoma group (G1,G2,G3)werecomparedandanalyzed.TheROCcurvewasemployedtoevaluatethediagnosticefficacyandthresholdof eachparameter.P earson correlation wasappliedtoanalyzethecorrelationbetweeneachparametervalueandpathologicalgrade.Results The MK valuesincreasedgradually,meanwhiletheMDandADCvaluesdecreasedgraduallyinG0,G1,G2andG3groups.ExceptforMDand ADCvaluesbetweenG0andG1groups,othervalueswerestatisticallysignificantdifferent(P<0.05)betweendifferentgroups.In differentiatingoftheG0/(G1+G2+G3),G0/G1,G1/G2,G2/G3,theMKvalueshadthehighestdiagnosticefficacy(AUC=09.2,07.6,09.0,0.96, P<0 .05 ).M oreover ,in the correlation of pathological grading ,M K>M D>A D C (r=0 .850 ,0 .781 ,0 .709 ,P<0 .05 ).Conclusion Both DKIandDWIcandiagnoseandevaluatepathologicalgradeofendometrialcarcinoma.ComparedwithDWI,DKIembracesmoreperfectmathematical modelandmoresensitiveparameters,andcanbeusedasaneffectivemethodtoevaluatethepathophysiologicalfeaturesofendometrialcarcinoma.

Objective To establish a HPLC method for the determination of related substances in lysine hydrochloride and zinc gluconate oral solution. Methods The HPLC separation was performed at 30 ℃ on an Inertsil Amide column (4.6 mm×250 mm,5 μm) by gradient elution with mobile phase A [phosphatesolution (2.72 g KH2PO4and 0.46 goctane sulfonate,added with 1 000 mL)-acetonitrile (22:78)] and B [phosphate solution-acetonitrile (95:5)] at the flow rate of 1.0 mL·min-1.The clumn temperature was 30 ℃.The determination wavelength was 203 nm. Results The major peaks were well separated from adjacent related substances peaks and resolution were greater than 2.0.The theoretical plate number of major peaks and related substance peaks were greater than 3 000.Excipients and destruction test products did not interfere with the determination. Conclusion This method is practicable,specific and durable for the simultaneous determination of related substances in lysine hydrochloride and zinc gluconate oral solution.

Objective:To investigate the effect of P2X4R silencing on 6-hydroxydopamine (6-OHDA) induced Parkinson's disease (PD) cell model and its mechanism. Methods:(1) According to the 6-OHDA concentrations, the SH-SY5Y cells were divided into 0 μmol/L 6-OHDA group, 50 μmol/L 6-OHDA group, 100 μmol/L 6-OHDA group, and 150 μmol/L 6-OHDA group. CCK-8 was used to detect the cell survival rate, Western blotting was used to detect the P2X4R protein expression, and real time quantitative RT-PCR was used to detect the P2X4R mRNA expression. The optimal concentration of 6-OHDA was selected to induce PD cell model. (2) SH-SY5Y cells at logarithmic phase were transfected with P2X4R siRNA lentiviral plasmids of different sequences (P2X4R-siRNA540, P2X4R-siRNA792, and P2X4R-siRNA1401) and nonsense sequence normal control plasmid (NC-siRNA), respectively (P2X4R-siRNA540 group, P2X4R-siRNA792 group, P2X4R-siRNA1401 group, and NC-siRNA group); the P2X4R mRNA and protein expressions were detected by real time quantitative RT-PCR and Western blotting, and the P2X4R siRNA sequence with the best silencing effect was screened to establish P2X4R silencing cell line. (3) The PD cells induced by the optimal concentration of 6-OHDA were transfected by P2X4R-siRNA enjoying the best silencing effect, NC-siRNA, and P2X4R antagonist CORM-2 (PD+P2X4R-siRNA group, PD+NC-siRNA group, and PD+CORM-2 group); CCK-8 and flow cytometry were used to detect the survival rate and apoptosis rate of cells in each group, and Western blotting was used to detect the protein expressions of connexin (PANX1), toll-like receptor (TLR)-2, Caspase-3 and cleaved Caspase-3. (4) The PANX1 or TLR-2 over-expression plasmids (pCMV3-PANX1 and pCMV3-TLR2), and negative control plasmid (pCMV3-NCV) were transfected into cells from the PD+P2X4R-siRNA group (PD+P2X4R-siRNA+pCMV3-PANX1 group, PD+P2X4R-siRNA+pCMV3-TLR2 group, and PD+P2X4R-siRNA+pCMV3-NCV group); CCK-8 and flow cytometry were used to detect the survival rate and apoptosis rate of cells in each group; Western blotting was used to detect the TLR-2, Caspase-3 and cleaved Caspase-3 protein expressions. Results:(1) As compared with that in the 0 μmol/L 6-OHDA group, the cell survival rate in 50, 100, and 150 μmol/L 6-OHDA groups was significantly decreased in a dose-dependent manner, and the P2X4R protein and mRNA expression levels were significantly increased in a dose-dependent manner ( P<0.05); among them, 100 mol/L 6-OHDA was the most suitable concentration to induce PD cell model. (2) As compared with those in the NC-siRNA group, the P2X4R mRNA and protein expressions in P2X4R-siRNA540 group, P2X4R-siRNA792 group, and P2X4R-siRNA1401 group were significantly decreased ( P<0.05); P2X4R mRNA and protein expressions were the lowest in the P2X4R-siRNA540 group. (3) As compared with PD+NC-siRNA group, the PD+P2X4R-siRNA group and PD+CORM-2 group had significantly increased survival rate, significantly decreased apoptosis rate, and statistically decreased Caspase-3, cleaved Caspase-3, PANX1 and TLR-2 protein expression levels ( P<0.05). (4) As compared with PD+P2X4R-siRNA+pCMV3-NCV group, the TLR2 protein expression in PD+P2X4R-siRNA+pCMV3-PANX1 group was significantly lower ( P<0.05); as compared with PD+P2X4R-siRNA+pCMV3-NCV group, PD+P2X4R-siRNA+pCMV3-TLR2 group had significantly increased cell survival rate, significantly decreased apoptosis rate, and significantly decreased Caspase-3 and cleaved Caspase-3 protein expressions ( P<0.05). Conclusion:P2X4R silencing can significantly improve the survival rate of PD cell model induced by 6-OHDA, reduce apoptosis and expressions of apoptosis related proteins (Caspase-3 and cleaved Caspase-3), and play a neuroprotective role, whose mechanism may be related to PANX1/TLR-2 signal pathway.