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

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

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 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 investigate the effect of creatine phosphate sodium on myocardial protection and calcium-sensitive receptor (CaSR) expression following high-level spinal cord injury.Methods Thirty healthy male SD rats weighing 250-300 g were assigned to sham operation,12-hour injury,24-hour injury,12-hour injury followed by a single intraperitoneal injection of creatine phosphate sodium,and 24-hour injury followed by a single intraperitoneal injection of creatine phosphate sodium according to the random number table,with 6 rats in each group.High-level spinal cord injury was induced at C7 segment by dropping a 10 g weight falling freely along the hollow glass tube from a 5 cm height.Level of blood troponin Ⅰ (cTnⅠ) was measured.Myocardial tissues were collected to study ultrastructure of myocardial cells under transmission electron microscope and CaSR expression using fluorescence quantitative PCR and Western blotting.Results cTnⅠ level was (0.031 ±0.002) U/L and (0.026 ± 0.001) U/L in 12-and 24-hour injury groups,but it was reduced to (0.023 ± 0.002) U/L and (0.018 ± 0.006) U/L at the same time point in treatment groups (P ＜ 0.05).Whereas either in injnry or treatment groups,cTnⅠ level was higher than (0.004 ± 0.002) U/L in sham operation group (P ＜ 0.05).CaSR mRNA level was (0.991 ±0.146) × 10-3 and (1.245 ±0.204) × 10-3 in 12-and 24-hour injury gronp and decreased to (0.880 ± 0.096) × 10-3 and (0.782 ± 0.138) × 10 3 at the same time point in treatment groups (P ＜ 0.05),but all were higher than (0.437 ± 0.065) × 10-3 in sham operation group (P ＜ 0.05).CaSR protein expressed in 12-and 24-hour injury group was (0.627 ±0.066) × 10 3 and (0.809 ±0.154) ×10 3 and lowered to (0.505 ±0.176) × 10-3 and (0.524 ±0.138) × 10-3 at the same time point in treatment groups,but all were higher than (0.331 ± 0.102) × 10-3 in sham operation group (P ＜ 0.05).Transmission electron microscopy demonstrated normal myocardial ultrastructure in sham operation group but impairment in injury groups,but the impairment was significantly improved in treatment groups.Conclusion Creatine phosphate sodium can decrease cTnⅠ level,attenuate the damage to myocardial ultrastructure and down-regulate CaSR after high-level spinal cord injury.

Objective To study etiology screening role of transrectal ultrasonography in male obstructive azoospermia infertility.Methods The clinical data of 328 cases who suspected of being obstructed sperm disease were retrospectively analyzed.TRUS detection was conducted,at the same time,the sperm amount,sperm and semen pH,pure berries quantitative,neutral sugar alpha glycosidase enzymes quantitative,elastic hard protease were tested.Results In 328 cases with male obstructed no sperm,by TRUS detection results,216 cases (65.8％) could find the causes,ejaculatory duct expansion,seminal vesicle gland lesions,prostate midline cyst were the top three causes respectively;112 patients(34.2％) had no obvious abnormal ultrasonic testing.Sperm was not seen in semen of obstructive azoospermia patients and semen pH ＜ 7,pure berries sugar quantitative and quantitative value neutral alpha glycosidase enzymes were very low,hard elastic protease was low.Conclusion The main causes of obstructive azoospermia were ejaculatory duct expansion,seminal vesicle gland lesions,prostate midline cyst,sperm TRUS detection used for diagnosis of high sensitivity,and easy to operate,noninvasive,and combined with seminal plasma biochemical examination,the diagnostic effect is much better.

Objective To analyze the variations of glycerol-3-phosphate dehydrogenase 1 like gene (GPD1-L) and address the association with sudden m anhood death syndrom e (SMDS). Methods The genom ic DNA was extracted from blood sam ples of the SMDS group and the norm alcontrolgroup.The exons, exon-in-tron boundaries and 3′-U TRs of coding region of GPD1-L w ere PCRam plified and DNAsequenced di-rectly to confirm the types of variations. The genotype frequency and allele frequency w ere analyzed statistically. Results There w ere tw ovariants in the SMDS group, c.465C>Tand c.*18G>T, the latter existed certain degree difference of genotype distribution and allele frequency betw een the SMDS group and the control group, but there was no statistically significant (P>0.05). Conclusion The relation be-tw een gene m utation of GPD1-L and the occurrence of Chinese SMDS deserves a further research.