OBJECTIVE: To construct eukaryotic expression plasmid of porcine CCK gene pIRES2-EGFP/CCK and express it in COS-7 cells and hamsters. Methods The aimed segments were obtained from intermediate vector pMD18-T/CCK and were inserted into an eukaryotic expression plasmid pIRES2-EGFP to construct a recombinant expression plasmid pIRES2-EGFP/CCK. The recombinant expression plasmid was transfected into COS-7 cells by liposome-mediated gene transfer method and was observed through fluorescence microscope. The plasmid was injected into the skeletal muscle of hamsters directly to detect the expression of the recombinant plasmid in vivo. RESULTS: A recombinant eukaryotic expression plasmid pIRES2-EGFP/CCK was successfully constructed. Green fluorescent protein could be detected in the transfected COS-7 cells 24, 48, and 72 hours after the transfection. On the 4th day postinjection into the skeletal muscle of hamsters, the protein could be detected at the injection site and the fluorescence intensity became much stronger on the 14th day than that on the 4th day. On the 42nd day the protein level increased. The green fluorescence protein was never expressed in the untransfected cells. CONCLUSION The porcine CCK gene eukaryotic expression plasmid pIRES2-EGFP/CCK is constructed successfully, and is expressed in mammal COS-7 cells and hamsters in vivo. The research paves the way for the cross immunity therapy of hamster pancreatic carcinoma.
Animals ; Base Sequence ; COS Cells ; Cancer Vaccines ; therapeutic use ; Chlorocebus aethiops ; Cholecystokinin ; biosynthesis ; genetics ; Cricetinae ; Eukaryotic Cells ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Muscle, Skeletal ; metabolism ; Pancreatic Neoplasms ; therapy ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Swine ; Transfection

A study on the microbial transformation of glycyrrhetinic acid (GA) was conducted by a fungus, Cunninghamella blakesleeana CGMCC 3.970 systematically. After incubation with the cell cultures of C. blakesleeana CGMCC 3.970 at 25 degrees C for 7 days on a rotary shaker operating at 135 r x min(-1), GA was converted into one major product and five minor products. The products were extracted and purified by solvent extraction, macroporous adsorbent resin, silica gel column chromatography, and semi-preparative RP-HPLC chromatography. Their structures were identified as 3-oxo-15α-hydroxy-18β-glycyrrhetinic acid(1), 3-oxo-15β-hydroxy-18β-glycyrrhetinic acid (2), 7β,15α-dihydroxy-18β-glycyrrhetinic acid (3), 3-oxo-7β, 15α-dihydroxy-18β-glycyrrhetinic acid (4), 7β-hydroxy-18β-glycyrrhetinic acid(5) and 15α-hydroxy-18β-glycyrrhetinic acid(6) by the analyses of MS, 1H-NMR and 13C-NMR spectroscopic data respectively. Among them, 2 was a new compound. These results suggest that C. blakesleeana CGMCC 3.970 has the capability of selective ketonization and hydroxylation for GA. [Key words] glycyrrhetinic acid; Cunninghamella blakesleeana CGMCC 3. 970; microbial transformation
Biotransformation ; Cunninghamella ; metabolism ; Glycyrrhetinic Acid ; analogs & derivatives ; chemistry ; metabolism ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization

Objective To collect the data of present basic constructive situation of intensive care units (ICUs) in second and tertiary grade hospitals in Xinjiang Uygur Autonomous Region and to provide a data base beneficial to the construction of ICUs in the whole region.Methods The information including basic situation of ICUs in a total of 49 second and tertiary grade hospitals as samples in Xinjiang Uygur Autonomous Region were investigated by questionnaire survey from December 2012 to April 2013. The contents of questionnaire chiefly included the basic situations of hospitals, ICU constructions, ICU human resources and allocation of equipments.Results Forty-nine questionnaires were sent out, and all of them gave answers, the recovery rate being 100%. In the whole region of Xinjiang, there were 49 second and tertiary grade hospitals with establishment of 66 various types of ICUs. ① Medical unit basic situation: the second grade A hospitals accounted for 59.2%, and the tertiary grade A hospitals, 34.7%. There was a larger proportion of local hospitals (67.3%); most of the hospitals had beds over 500 (57.14%), the designed hospital bed number was 41 403, and 35 442 beds were open up for service (85.60%). ② The ICU basic construction situation: the construction of ICUs concentrated mainly after the year 2000, from 2000 to 2010, a total of 37 units, and from 2010 to 2012, 12 units were constructed; when they opened to the public, the beds were relatively few, and the ICUs containing less than 10 beds occupied 79.00%. In the 66 ICUs surveyed, there were 43 general ICUs, accounting the largest proportion, followed by 7 emergency ICUs, and the least were 1 neurosurgery and 1 cardiac surgery ICU. In the 66 ICUs, all together 765 beds were prepared, but actually 808 beds were opening up to public; most of ICUs had 5 - 20 beds, accounting for 71% in all the ICUs. At present, in the whole Xinjiang region, the rate of beds in ICUs in various grades of hospitals opening to the outside for use accounted for more than 80% of which 56% ICUs were almost turning round fully. ③ ICU human resources situation: totally, there were 484 doctors in ICUs in the whole region, the ratio of full-time doctor to nurses was 1:3.50, the ratio for bed to physician 1:0.55 and the ratio for bed to nurse 1:0.92. The title of doctor was primarily resident, and the various title gradient levels were good. Most doctors graduated from a regular medical college, and the doctors having obtained a master degree were few. The physician professional backgrounds were mainly critical disease medicine, emergency medicine and respiratory medicine, the professional relevance being good; the chief way of ICU physicians to engage in advanced study of their professional training was in China, and generally they joined professional continuing education programs poorly, mostly joining once a year or non at all. The physicians who obtained the identification of Chinese critical care medicine specialist accounted only for 23.56%. ④ ICU equipment allocation situation: according to the requirements of ICU construction guidelines, the equipments must be allocated including bedside monitors (703 units), ventilators (516 units) and infusion pumps (702 units), occupying the highest proportion. In the optional equipments, there were enteral nutritional pump (89 stations), blood glucose monitoring device (57 units) and anti-decubitus mattresses (54 units), the proportion being the largest, bispectral index monitor (2 units), extracorporeal membrane oxygenation (ECMO, 1 table) and gastric mucosal carbon dioxide tension pHi meter (0 unit), the proportion being the least or non at all.Conclusions In the whole Xinjiang region, the construction of ICUs is still at the developing stage, and needs to further strengthen the standardized construction. The human resources, equipments, etc. are not distributed in balance, and it is necessary to move forward the adjustment to benefit the development of ICUs in the whole region.

Actins ; metabolism ; Bone Neoplasms ; blood ; complications ; diagnostic imaging ; pathology ; surgery ; Diagnosis, Differential ; Female ; Humans ; Hypophosphatemia ; blood ; etiology ; Ilium ; Mesenchymoma ; blood ; complications ; diagnostic imaging ; pathology ; surgery ; Middle Aged ; Osteomalacia ; blood ; etiology ; Phosphates ; blood ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Tomography, X-Ray Computed

Objective: To investigate the therapeutic mechanism of Tripterygium polyglucoside (TⅡ) in treating asthma patients. Methods: Thirty asthma patients of middle or sever leoel were selected and randomly divided into three groups, Group A treated with TⅡ, Group B treated with prednisone and Group C, the control group. Their peripheral CD４＋, CD8＋ T-lymphocytes were counted by flow cytometer and serum interleukin-5 (IL-5) level determined by ELISA before and after 4 weeks′ treatment. Results: In Group A, after treatment, CD４＋ T-lgmphocytes reduced from 0.462±0.035 to 0.426±0.039 (P＜0.01), CD8＋ increased from 0.201±0.045 to 0.253±0.043 (P＜0.01), and serum IL-5 median concentration reduced from 65.3 ng/L to 10.9 ng/L (P＜0.01). Similar results was revealed in Group B, while there was insignificant change of the parameters in Group C. The serum concentration of IL-5 was positively correlated with the peripheral CD４＋ count (r＝0.61, P＜0.01). Conclusion: TⅡ is highly effective in treating asthma through regulating T-lymphocyte subset disorder and inhibiting production of IL-5.

Aged ; Humans ; Male ; Mesothelioma ; diagnosis ; pathology ; Peritoneal Neoplasms ; diagnosis ; pathology

ObjectiveTo investigate the iodine nutritional status of pregnant women,newborn heel blood thyroid stimulating hormone(TSH) level and their relationship with urinary iodine(UI) level during pregnancy in Zhoupu and Kangqiao districts of Pudong New Area of Shanghai.Methods A total of 993 urinary samples(the first,second and third trimesters of pregnancy were 200 people,respectively),breast feeding(193 people) and non-pregnant women (200 people) in Zhoupu and Kangqiao districts of Pudong New area were collected from Apr 2009 to Dec 2010.Two hundred copies of neonatal heel blood samples were collected.Median of UI was measured by arsenic-cerium catalysis.TSH in neonatal heel blood was analyzed 72 h after birth by time resolved fluoroisnmunoassay(TRFIA).ResultsMedian UI of all pregnant women was 161.35 μg/L,and that in third trimesters of pregnancy( 126.35 μg/L) was lower than that of the first,the second,the breast feeding and non-pregnant women (178.80,180.50,167.90,163.40 μg/L,all P＜ 0.05).The percentage of UI level less than 150 μg/L in the third trimester[57.5％(115/200) ] was higher than that of the first[39.0％(78/200) ],the second[39.5％(79/200) ],the breast feeding [ 16.6％ (32/193) ] and non-pregnant women [ 23.0％ (46/200) ],respectively (all P ＜ 0.05).The percentage of UI level higher than 300 μg/L in the first [9.0％(18/200)],the second[8.0％(16/200) ] and the third trimester [ 5.0％ ( 10/200 ) ] of pregnancy was lower than that of the breast feeding [ 20.2％ (39/193) ] and nonpregnant [20.5％(41/200) ] women,respectively(all P ＜ 0.05).The level of neonatal heel blood TSH was(2.92 ± 1.83)mU/L,the range was 0.01 - 9.76 mU/L,11.0％(22/200) of the neonates heel blood TSH level(5 mU/L)exceeded the ratio of World Health Organization (WHO) standard ( ＜ 3％ ) suitable for iodine nutrition.Conclusions The overall level of iodine nutrition among pregnant women in Zhoupu and Kangqiao districts of Pudong New Area of Shanghai is in the appropriate range,but the pregnant women in the third trimester is in mild iodine deficiencies,and the neonates in these districts may be prone to iodine deficiency.Monitoring of iodine nutrition of pregnant women should be strengthened and iodine supplementation should be done scientifically.

OBJECTIVETo observe the intervention and mechanism of Qushi Huayu Recipe (QHR) on gene expression profiles in high lipid diet induced fatty liver rats.

METHODSFatty liver model was prepared in 20 male SD rats using single high fat diet (88% common forage +2% cholesterol +10% lard). Four weeks after modeling they were divided into the model group and the QHR group according to random digit table, 10 in each group. QHR (at 0. 93 g crude drug/100 g body weight) and distilled water was respectively to rats in the QHR group and the model group by gastrogavage while modeling, once per day. Meanwhile, 10 SD male rats were recruited in a normal group, administered with equal volume of distilled water by gastrogavage. At the end of week 8 all rats were sacrificed, and blood and livers were collected for subsequent analysis. Contents of liver triglyceride (TG) and free fatty acid (FFA) , activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using biochemical assay. Pathological changes of liver tissue were observed using H&E and oil red O stain. Liver gene expressions were detected by Affymetrix gene expression profiles. Differentially expressed genes were compared between the QHR group and the model group, functions of differentially expressed genes and signal pathways involved analyzed. Ten differentially expressed genes involved in glycolipid metabolism with fold change more than 2 were selected for verification by real-time PCR.

RESULTS(1) Compared with the normal group, contents of liver TG and FFA, and serum activities of ALT and AST obviously increased in the model group (P <0. 01). Compared with the model group, contents of liver TG and FFA, and activities of ALT and AST obviously decreased in the QHR group (P <0. 05, P <0. 01). QHR could reduce high fat induced fatty degeneration of liver cells , alleviate inflammation, and improve pathological changes of liver tissue. (2) Compared with the model group, there were 80 differentially expressed genes (with fold change > 2, P < 0.05) with clear functions and appointed gene names, including 44 up-regulated and 36 down-regulated genes. Eighty genes were involved in 27 signal pathways with statistical difference, including glycerolipid metabolism, adipocytokine signaling pathway, insulin signal pathway, drug metabolism signal pathway, etc (P < 0.05). (3) RT-PCR results of 10 glycolipids metabolism regulating genes such as Gk, Scd1, Gpat2, G6pc, Irs1, and so on showed that all RT-PCR genes were completely coincide with up-regulated or down-regulated tendency in results of gene chips. 80% genes had approximate fold change.

CONCLUSIONQHR could regulate gene expressions related to fat metabolism, carbohydrate metabolism, anti-lipid peroxidation, and drug metabolism in high fat diet induced fatty liver rats, and its comprehensive pharmacological actions could be manifested.

Alanine Transaminase ; metabolism ; Animals ; Aspartate Aminotransferases ; metabolism ; Carbohydrate Metabolism ; Diet, High-Fat ; Drugs, Chinese Herbal ; pharmacology ; Fatty Acids, Nonesterified ; metabolism ; Fatty Liver ; metabolism ; Lipid Metabolism ; Lipid Peroxidation ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Transcriptome ; drug effects ; Triglycerides ; metabolism

This paper reports the establishment of a method for rapid identification 15 effective components of anti common cold medicine (paracetamol, aminophenazone, pseudoephedrine hydrochloride, methylephedrine hydrochloride, caffeine, amantadine hydrochloride, phenazone, guaifenesin, chlorphenamine maleate, dextromethorphen hydrobromide, diphenhydramine hydrochloride, promethazine hydrochloride, propyphenazone, benorilate and diclofenac sodium) with MRM by LC-MS/MS. The samples were extracted by methanol and were separated from a Altantis T3 column within 15 min with a gradient of acetonitrile-ammonium acetate (containing 0.25% glacial acetic acid), a tandem quadrupole mass spectrometer equipped with electrospray ionization source (ESI) was used in positive ion mode, and multiple reaction monitoring (MRM) was performed for qualitative analysis of these compounds. The minimum detectable quantity were 0.33-2.5 microg x kg(-1) of the 15 compounds. The method is simple, accurate and with good reproducibility for rapid identification many components in the same chromatographic condition, and provides a reference for qualitative analysis illegally added chemicals in anti common cold medicine.
Acetaminophen ; analysis ; Acetanilides ; analysis ; Amantadine ; analysis ; Aminopyrine ; analysis ; Anti-Inflammatory Agents, Non-Steroidal ; analysis ; Antipyretics ; analysis ; Antipyrine ; analogs & derivatives ; analysis ; Caffeine ; analysis ; Chlorpheniramine ; analysis ; Chromatography, Liquid ; Diclofenac ; analysis ; Diphenhydramine ; analysis ; Drug Contamination ; Drug Stability ; Ephedrine ; analogs & derivatives ; analysis ; Guaifenesin ; analysis ; Promethazine ; analysis ; Pseudoephedrine ; analysis ; Reproducibility of Results ; Salicylates ; analysis ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry

Objective To compare the clinical results of anterior cruciate ligament(ACL)reconstruction using anterior tibial muscle tendons allograft and hamstring tendons autograft.Methods According to the graft used for ACL reconstruction,60 patients with chronic ruptures of ACL were divided into group A(30 patients,using anterior tibial muscle tendons allograft)and group B(30 patients,using semitendinosus and gracilis tendons autograft).The average follow-up periods after surgery were 29.4 and 31.6 months respectively.Lysholm,Tegner,IKDC clinical rating scales and KT2000 were used for evaluation pre-and post-operation.The muscle strength of the extension,flexion,internal rotation and external rotation of knees was evaluated by the isokinetic tester.Results There were statistically significant differences in Lysholm,Tegner,IKDC clinical rating scales and KT2000 results between pre-and postoperation(P<0.01).However,there were no statistically significant differences between group A and group B after operation(P>0.05).The Isokinetic muscle strength tester showed that the knee muscle strength of the internal rotation and flexion were decreased post-operative in group B,rather than group A,which showed no significant differences between pre-and post.operation(P<0.05).Conclusion For ACL reconstruction under arthroscopy,anterior tibial muscle tendons allograft was a favorable substitute for autograft materials for its convenience,satisfactory clinical effect and less complications.