Objective:To establish a liquid chromatography-mass spectrometry(LC-MS) method for determining the concentration of amlodipine besylate in human plasma and to evaluate the bioequivalence of 2 kinds of amlodipine besylate tablets.Methods: Twenty healthy male volunteers were enrolled into a single crossover study.A single dose of the suspension equivalent to 10 mg amlodipine besylate or a reference preparation was given in a crossover way.The plasma concentrations of amlodipine besylate were determined by LC-MS method in the volunteers at different time points;the pharmacokinetic parameters and relative bioavailability were calculated and the bioequivalence of the 2 preparations were evaluated.Results: The pharmacokinetic parameters for experimental and the reference preparations were: C_max(6.21?1.88) vs(6.03?1.08) ng/ml;AUC_0-120(250.68?52.61) vs(246.14?52.11) ng h/ml;T_max(6.0?2.3) vs(6.1? 2.5) h;t_1/2(40.45?6.68) vs(43.74?9.05) h,respectively.The linear range of the present method was 0.1-20.0 ng/ml;the lowest detectable concentration of amlodipine besylate was 0.1 ng/ml.There was no significant difference in pharmacokinetic parameters between the 2 tablets.Conclusion: The present method is simple to use,fast,and accurate.The 2 preparations of amlodipine besylate are bioequivalent.

BACKGROUND:As current studies on isolation, culture andcryopreservation of human amniotic epithelial cells (hAECs) are relatively scattered, it is difficult to form a comprehensive and effective solution to meet the clinical needs of stem cells for transplantation in future.OBJECTIVE:To establish the technology of isolation, culture and cryopreservation of hAECs, and to study the biological characteristics of hAECs.METHODS:Orthogonal method was used to study the effects of different factors on the separation, culture and cryopreservation, and range method was adopted to analyze the data to optimize the separation, culture and cryopreservation. We performed cell primary and passage cultures, morphology observed by microscope, drawn cell growth curve and flow cytometry assay, immunofluorescence staining, hepatocyte like cell differentiation to study the biological characteristics of hAECs.RESULTS AND CONCLUSION:(1) The optimal hAECs separation conditions were as follows:trypsin digestions were conducted at a concentration of 0.25%, four times, once for 20 minutes digestion; optimal conditions of culture were 4×108/L cell seeding density, 10 μg/L epidermal growth factor, 5% serum; optimal conditions of cryopreservation were 1×1010/L cell cryopreservation density, 10% dimethyl sulfoxide, 80% serum. (2) The primary cells were adhered to the wall in 2-3 days, exhibiting irregular polygon, paving stone-like growth. Cell adherence and growth rate were accelerated after subculture, and the growth and proliferation ability of passage 2 cells were not significantly decreased after cryopreservation and resuscitation. (3) Immunofluorescence staining showed that the primary cells strongly expressed SSEA-4 and CK19, but did not express Vimentin, CD45 and HLA-DR. The immunophenotype statistics of the primary and passage 4 cells showed the epithelial mesenchymal transition of hAECs in culture process. (4) Immunofluorescence staining showed that the liver cell marker expression of ALB, CK18 was significantly increased after hAECs were induced to differentiate into hepatocyte-like cells. Glycogen staining revealed glycogen synthesis in hAECs after 3 weeks of induction. To conclude, hAECs are easy to obtain and have strong proliferation ability in vitro, and express surface markers for undifferentiated embryonic stem cells.

Heparinase II (Hep II) from Flavobacterium heparinum is an enzyme that could specifically cleave certain sequence of heparin and heparan sulfate. In this work, fermentation conditions of recombinant heparinase II (His-Hep II) producing bacteria were optimized, including initial induction time, inducer (IPTG) concentration, induction temperature and induction time. The optimum conditions were as follows: cultivating recombinant bacteria to exponential prophase under 37 degrees C, then adding IPTG to a final concentration of 0.3 g/L, finally cultivating recombinant bacteria under 20 degrees C for 10 h. The total crude enzyme activity reached 570 U/L. Based on these results, high cell density fermentation of recombinant bacteria was studied. The final OD600 could reach 98 and the total crude enzyme activity of His-Hep II increased to 9 436 U/L.
Fermentation ; Flavobacterium ; metabolism ; Microbiological Techniques ; Polysaccharide-Lyases ; biosynthesis ; Recombinant Proteins ; biosynthesis

Objective To explore a new method of functional reconstruction of hand digits and joints with free transfer of foot tissues so as to increase the success rate of the operation.Methods After micro-anatomic study of the plantar and dorsal metatarsal arteries,retrograde and free grafts of foot tissues pedicled with plantar metatarsal arteries were designed and applied in transplantation to treat 76 cases of hand digital or joint defects.The surgeries included 58 cases of transfer of the second toe,four cases of transfer of composite tissues of the second toe, eight cases of transfer of proximal interphalangeal joint,and six cases of nail flap transfer.Results The mi- cro-anatomic study found that the first plantar metatarsal artery was anatomically constant and the diameter of its branch to the second toe was larger than that of the first dorsal metatarsal artery.Flaps survived in 75 of the 76 patients(98.7%),with fine appearance and significantly improved function.One patient who had received free transfer of the second toe to reconstruct the thumb function had to undergo a second repair with infraclavicula skin tube because of refractory arteriospasm of anastomosed vessels.Conclusion Transfer with free retrograde grafts of foot tissues pedicled with plantar metatarsal artery to reconstruct hand functions can effectively improve the success rate of the operation,because it is free of the shortcomings of great anatomic variation of blood vessels and time-consuming and complex procedures in conventional transfer.

A rapid LAMP detection method with primers designed on genus-specific region identified in the gyrA gene was established in this assay. All four Campylobacter jejuni from different sources were detected positive and fourteen non Campylobacter bacteria were negative, which shows excellent specificity of the primers. Compared with plate count and PCR method, the LAMP method and the PCR method had equal sensitivity, which were three orders of magnitude higher than plate count. In this assay, we also found out that the treatment of DNase could reduce the dead bacteria DNA effectively. The LAMP detection on chicken indicated relatively good result on detection of Campylobacter jejuni combining with treatment of DNase.

Objective To induce mouse embryonic stem(ES)cells to differentiate into insulin-secreting cells by means of a 5-step model system.Methods E14.1 mouse ES cells were cultured in the presence of leukemia inhibitory factor(LIF)for 2 days(step 1),then the cells were cultured in hanging drops to form embryonic bodies(EBs)and the resulting EBs were cultured in suspension for 6 days in the presence of basic fibroblast growth factor bFGF(step 2).Subsequently the EBs were cultured in the medium containing glucagon- like peptide 1(GLP-1),hepatocyte growth factor(HGF),nerve growth factor(NGF)and nicotinamide for 10 days(step 3).After that,the EBs were dissociated into single cells,and the cells were cultured in monolayer in the presence of GLP-1,betacellulin,activin A,bFGF and nicotinamide for 10 days(step 4).Finally,the cells were cultured in low-glucose medium containing nicotinamide for 4 days(step 5).Insulin and some other islet- related genes expressions were investigated using RT-PCR and insulin expression was also investigated by DTZ- staining and immunohistochemistry.The percentage of insulin-secreting cells was evaluated by flowcytometry and insulin concentrations were measured by RIA.Results mRNA expression of insulin became visible at step 3 and more evident at step 5.Additionally,at step 5,mRNAs of glucagon,somatostatin,pancreatic polypeptide(PP), pancreatic duodenal homeobox 1(PDX-1),beta-cell E box transactivator 2(Beta2)and neurogenin 3(Ngn3) were detected.DTZ-staining positive cells and insulin immunohistochemical staining positive cells were observed. The percentage of insulin-positive cells was(24.0?2.5)%(n=6).In the presence of 5.6 mmol/L and 25 mmol/L glucose,insulin concentrations were(0.05?0.01)?g/L and(0.13?0.02)?g/L respectively(n= 6).Conclusion E14.1 mouse ES cells can be induced to differentiate into insulin-secreting cells by the 5-step model system.Insulin-secreting cells can release insulin into culture medium when treated with glucose,and insulin concentrations increase with rising concentration of glucose.

Objective To evaluate the clinical value of 131 Ⅰ SPECT/CT in the differentiation of indeterminate 131Ⅰ uptake on planer whole body scan (WBS) for patients with DTC after 131Ⅰ treatment. Methods Fifty-six DTC patients ( male: 19, female: 37, mean age: 45 ± 15 years, ranging from 20 to 85 years) underwent 131Ⅰ treatment. 131Ⅰ WBS was performed five days after 131 Ⅰ treatment, followed by regional 131Ⅰ SPECT/CT for the indeterminate foci with abnormal uptake on 131Ⅰ WBS. The diagnostic difference of the two imaging modalities was compared by x2 test. Results There were 288 foci with abnormal uptake on 131 Ⅰ WBS, including 108 indeterminate foci (37.5%). Subsequent 131Ⅰ SPECT/CT identified 27 foci as DTC metastases (25.0%) and 71 foci as non-metastases such as benign lesions at nose, oral cavity, salivary gland, maxillary cyst, thyroid remnant, thymus, gallbladder, gastrointestinal tract, and uterus, or non-specific uptake of body contaminations (65.7%). However, the remaining 10 foci (9.3%) remained indeterminate on 131 Ⅰ SPECT/CT imaging. The diagnostic accuracy of 131 Ⅰ SPECT/CT was significantly higher than that of 131Ⅰ WBS (x2 = 102.35, P＜0. 01). Conclusion 131Ⅰ SPECT/CT could significantly improve the diagnostic accuracy for the differentiation of indeterminate foci with abnormal uptake on 131Ⅰ WBS.

OBJECTIVEA study was carried out to investigate the effects of seed oil and sarcocarp oil of Hippophae rhamnoides on rats with experimental hepatocirrhosis, and comparison between the two.

METHODA rat model of experimental hepatocirrhosis was set up by feeding CCl4. Different concentration of seed oil and sarcocarp oil of H. rhamnoides were feed to those rats for 45 d, then the changes of activity of ALT in serum and SOD in liver were measured.

RESULTBoth of seed oil and sarcocarp oil can control the increase of ALT in serum and the decrease of SOD evidently, and the effect of seed oil was turn out to be a little better than sarcocarp oil.

CONCLUSIONSeed oil was more effective than sarcocarp oil of H. rhamnoides in alleviating liver injury caused by CCl4.

Alanine Transaminase ; blood ; Animals ; Carbon Tetrachloride Poisoning ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Fruit ; chemistry ; Hippophae ; chemistry ; Liver ; enzymology ; Liver Cirrhosis, Experimental ; etiology ; metabolism ; Male ; Plant Oils ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; Rats ; Seeds ; chemistry ; Superoxide Dismutase ; metabolism

Aneurysmal subarachnoid hemorrhage accounted for 70% of subarachnoid hemorrhage. The high fatality and disability rate affected patients′ quality of life. Aneurysmal subarachnoid hemorrhage should be considered as a chronic cerebrovascular disease, and it is very important to take early intervention and strengthen the secondary prevention to the patients with risk factors for disease. This paper reviewed the research status on quality of life of patients with aneurysmal subarachnoid hemorrhage and its influencing factors, in order to provide a reference for neurological physicians.

Objective This study was designed to observe the impact of cigarette cessation on anxiety and depression in patients with coronary heart disease (CHD).Methods A total of 690 cigarette smoking patients with CHD identified by coronary angiography (CAG) were included and analyzed in the study.The mental state were scored with Hamilton anxiety (HAMA) and depression (HAMD) scale both on admission and at 6-month follow-up.The patients were divided into two groups based on the cigarette cessation.The score of mental state between the two groups were compared.The patients were treated with percutaneous coronary intervention (PCI),coronary artery bypass grafting (CABG),or medicine therapy (MT).Results The clinic data and score of mental state were similar at the time of admission (HAMA:10.66±5.53 vs 11.09 ±5.61,P =0.311;HAMD:29.81 ± 10.13 vs 28.94 ± 10.22,P =0.266 4) between the two groups.After 6 months,the proportions of subjects in smoking cession group with anxiety (24.2％ vs 32.3％,P ＜0.05),depression (18.0％ vs 27.5％,P ＜0.05),and anxiety and depression (7.0％ vs 16.2％,P ＜0.001) decreased significantly compared with those in smoking group irrespective of the treatment strategy.Both the HAMA and HAMD scores were lower in smoking cessation group (HAMA:9.83±3.40;HAMD:24.91 ±7.90) than in smoking group (HAMA:10.98 ±4.87;HAMD:27.70 ± 11.16) (all P ＜ 0.001).Conclusions Smoking cessation is good for the relief of anxiety and depression in CHD patient.