OBJECTIVE: To discuss blood collection tubes with different additives and their effects on the testing results of alcohol concentration in blood samples. METHODS: Blood samples from 10 volunteers were collected 2 hours after drinking with seven different types of disposable vacuum blood collection tubes, including ordinary tube without anticoagulant, coagulant tube, separating gel-coagulant tube, sodium citrate (1:4) tube, sodium citrate (1:9) tube, sodium citrate (9:1) tube and EDTA-K2 tube. The alcohol concentrations in these blood samples were analyzed by headspace gas chromatography. RESULTS: The concentration testing results of the same blood samples in different types of tubes were different from one to another. The sequence was as follows: separating gel-coagulant tube > coagulant tube > ordi- nary tube without anticoagulant > EDTA-K2 tube> sodium citrate (1:9) tube> sodium citrate (1:4) tube, whereas the results of the same blood sample in sodium citrate (1:9) tube and sodium citrate (9:1) tube showed no obvious difference. CONCLUSION It is better to collect a suspicious drunk driver's blood sam- ple using a disposable vacuum blood collection tube, with the EDTA-K2 tube being preferred.
Anticoagulants ; Blood Specimen Collection/methods* ; Citrates ; Ethanol/blood* ; Humans ; Sodium Citrate

Objective To establish luciferase-labeled mesenchymal stem cells in vitro.Methods From May,2013 to January,2014,recombinant lentiviral vectors containing luciferase gene was transfected in to mesenchymal stem cell line C3H10T1/2.Puromycin was used to filter the cells.Western-blot and immunofluorescenceimaging were used to detect the transfection efficiency.Results Western blotting analysis showed that the size of this protein in cells expressing about 60KD.Showed that infected C3H10T1/2 cells expressing luciferase protein.I mmunofluorescence,compared with the uninfected control group,virus-infected cells group of about 100％ of the cells were positive for red fluorescence.Show that had built a good stable expression of luciferase C3H10T1/2 cells.Conclusion A monoclonal cell line stably and highly expressing luciferase is obtained with luciferase-labeled mesen chymal stem cells.

Beta-Elemene is an antitumor drug which is isolated from the traditional Chinese medicinal herb Curcumae Phaeocaulis Rhizoma, it is the main component of elemene which is extracted from the plant and delivered via blood circulation after intravenous injection. The antitumor effect of beta-elemene in vitro and in vivo was definite, and beta-elemene could improve the patient immunity and no sever side effect, drug resistance or bone marrow suppression were found during the clinical studies. And human serum albumin (HSA) is a primary extracellular protein which has a high concentration distribution in blood plasma and has many characteristic physiological functions. Therefore, the binding of beta-elemene to protein may be very important for absorption, distribution, metabolism and elimination. Therefore, the study on the interaction of beta-elemene with drug-carrying protein is very important. In this work, molecular binding of beta-elemene to human serum albumin (HSA) was investigated by using spectrofluorometer. the binding constants suggested that a strong interaction and the formation of a complex between beta-elemene and HSA. This clearly implies that beta-elemene can be stored and removed by the proteins in the body. Furthermore, the fluorescence quenching results showed that the HSA fluorescence was quenched by beta-elemene through static quenching mechanism. Thermodynamic parameters showed that hydrophobic interactions play a role in the binding of beta-elemene to HSA. The negative deltaH(0) and positive deltaS(0) in case of beta-elemene therefore showed that electrostatic attraction play a role in the binding of beta-elemene to HSA.
Drugs, Chinese Herbal ; chemistry ; Humans ; Kinetics ; Protein Binding ; Serum Albumin ; chemistry ; Sesquiterpenes ; chemistry ; Spectrometry, Fluorescence ; Thermodynamics

OBJECTIVETo investigate the effect of gastrodia in extracorporeal oriented inducing the differentiation of mesenchymal stem cells into neuron-like cells.

METHODSMesenchymal stem cells were separated from bone marrow of rats by wall sticking method, amplifying cultured in vitro, and differentiated into neuron-like cells by oriented induction with gastrodia. The morphology of cells was observed under light microscopy, neuro-specific enolase (NSE), nestin and glial fibrillary acidic protein (GFAP) were detected by immunocytochemistry.

RESULTSRats mesenchymal stem cells could be separated and amplified in vitro. After being induced by gastrodia for 2 hrs, most of the cells would be differentiated into meuron-like cells, revealing cytodendrite. By immunochemical staining, cells showed positive of NSE, nestin, and negative of GFAP.

CONCLUSIONRats' mesenchymal stem cells could be induced to differentiate into neuron-like cells.

Animals ; Animals, Newborn ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Gastrodia ; Glial Fibrillary Acidic Protein ; metabolism ; Intermediate Filament Proteins ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neurons ; cytology ; Phosphopyruvate Hydratase ; metabolism ; Rats ; Rats, Sprague-Dawley

Objective To detect the levels of cytokine IL-2,IL-12,IFN-?and IL-4 secreted by peripheral CD8~+ T lymphocytes in patients with condyloma acuminatum (CA).Methods Flow cytometry was employed to study the expression of cytokines IL-2,-12,INF-?and IL-4 in CD8~+ T lymphocytes in the peripheral blood of 60 patients with CA and 20 healthy controls.Results The percentage of CD8~+ T lym- phocytes producing IL-2,IL-12 and IFN-?were significantly lower in CA patients than that in healthy con- trols (P

OBJECTIVEEffects of ginsenoside Rb1, Rg1 and Re on neurotrophic factor signal transduction pathway using liposome-mediated transfection of eukaryotic cells approach.

METHODThe injury model was established by treating SH-SY5Y cells with 0.6 mmol x L(-1) of corticosterone (CORT) by 24 h. SH-SY5Y cell were pretreated with CORT for 30 min followed by co-treated with 120,60 and 20 micromol x L(-1) of Rb1, 120, 80 and 40 micromol x L(-1) of Rg1 and 120, 80 and 40 micromol x L(-1) of Re for 24 h. Cells viability was determined by Cell Counting Kit (CCK) assay. CREB expressing Luciferase reporter gene was constructed and transfected with plasmid containing hRaf, hcAMP, hAkt, hCaMK gene into human embryonic kidney (HEK293) cells using liposornal transfection reagent lipofection 2000. The expression of CREB before and after it addion of Rb1, Rg1 and Re was examined by Luc assay system and Western blotting.

RESULTCompared with normal control group, CORT significantly decreased the viability of SH-SY5Y cells to 67.21% (P < 0.01). CCK results show that Rb1 (60 micromol x L(-1)), Rg1 (80 micromol x L(-1)) and Re (80 micromol x L(-1)) on SH-SY5Y cells have significant protective effect (P < 0.01). Lucassay and Western blotting results show that the gene and protein levels of CREB increased significantly through the pathway of Raf and Akt with Rb1 and Rg1 (P < 0.01), Re can increase significantly the gene and protein levels of CREB through the pathway of Raf and CaMK II.

CONCLUSIONRb1, Rg1 and Re protects SH-SY5Y cells from CORT-induced damage and the neuroprotective mechanism may be associated with the Raf-CREB, Akt-CREB and CaMK II -CREB pathways.

Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; genetics ; metabolism ; Cell Line ; Cell Survival ; drug effects ; Cyclic AMP Response Element-Binding Protein ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Genes, Reporter ; Ginsenosides ; pharmacology ; Humans ; Panax ; chemistry ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Signal Transduction ; drug effects ; raf Kinases ; genetics ; metabolism

Five compounds (tenuifoliside C, tenuifoliside D, telephiose A, telephiose C and polygalaxanthone III) from polygala tenuifolia wild were incubated together with CYP probe substrate in human liver microsomes to investigate the inhibitory effect towards CYP450 enzyme. Phenacetin (CYP1A2), coumarin (CYP2A6), paclitaxel (CYP2C8), diclofenac (CYP2C9), S-mepheriytoin (CYP2C19), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1), midazolam (CYP3A) were selected as the isoforfn specific substrate. And the formation of paracetamol, 7-hydroxycoumarin, 6alpha-hydroxy paclitaxel, 4'-hydroxydiclofenac, dextrorphan, 6-hydroxychlorzoxazone, 1'-hydroxymidazolam, 4'-hydroxymephenytoin were detected respectively to measure the effect towards CYP450 by high-pressure liquid chromatography (HPLC). The result shows that five compounds from polygala tenuifolia willd significantly inhibit chlorzoxazone 6-hydroxylation catalyzed by CYP2E1, while showed no effect towards CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A. And IC50 value was 38.73, 54.14, 61.77, 62.22, 50.56 micromol x L(-1), respectively.
Cytochrome P-450 Enzyme System ; metabolism ; Esters ; pharmacology ; Glycosides ; pharmacology ; Humans ; Microsomes, Liver ; drug effects ; enzymology ; Oligosaccharides ; pharmacology ; Polygala ; chemistry ; Xanthones ; pharmacology

OBJECTIVETo observe the application effect of minimally invasive decompression, bone graft implantation and metal trabecular bone reconstruction system for early stage osteonecrosis of femoral head and discuss the treatment of hip-salvage operation in early stage osteonecrosis of femoral head;

METHODSFrom January 2010 to June 2011, 50 patients (62 hips) Which were osteonecrosis of femoral head of early stake,were treated with minimally invasive decompression, bone graft implantation and metal trabecular bone reconstruction system, including 31 males (40 hips), 19 females (22 hip) with an average age of 36.2 years old ranging from 22 to 54 years old. The course of disease was from 6 to 15 months (averaged 10.5 months). Among them, 19 cases (23 hips) were steroid-induced, 25 cases (33 hips) were alcohol-induced, 6 cases (6 hips) were idiopathic; According to ARCO stage, 28 hips were at stage I, 34 hips were at stage II. All of them were diagnosed as femoral head necrosis by imaging examination before operation. Then each patient was followed to assess by Harris hip score, curative effect, and conduct the femoral head survival analysis during the postoperation.

RESULTSAll patients had finished operation, the operation time was between 30 and 85 min, intraoperative blood loss was 50 to 220 ml, and 47 cases (58 hips) were follow-up from 24 to 46 months with an average of 34.05 months. As compared with preoperative, the Harris hip score at the last follow-up was improved, the difference was statistically significant (P<0.01). The Harris hip score, curative effect and survival time of femoral head in ARCO stage I was superior to these in ARCO Stage II, the difference was statistically significant (P< 0.05).

CONCLUSIONEffect of minimally invasive decompression,bone graft implantation combine with the metal trabecular bone reconstruction system for early stage osteonecrosis of femoral head was good,it could significantly improve the Harris hip score, increase the femoral head survival time, delay the hip replacement, and performance better in ARCO stage I.

Adult ; Bone Transplantation ; Decompression, Surgical ; Female ; Femur Head ; injuries ; pathology ; surgery ; Femur Head Necrosis ; surgery ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Prostheses and Implants ; Reconstructive Surgical Procedures ; Young Adult

Dingzhi Xiaowan is a widely used traditional Chinese medicine in treating depression, which is a similar formula of Kaixinsan. In this research, a rapid ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS(E)) method was established to analyze the metabolites of Dingzhi Xiaowan in depressive model rat plasma, bile, urine and feces. After we established Chronic unpredictable mild stress (CUMS) model rats and orally administrated Dingzhi Xiaowan, rat plasma, bile, urine and feces samples were collected and prepared. Using Waters Cortects UPLC C18 column (2.1 mm x 50 mm, 1.6 μm), acetonitrile-0.1% formic acid mobile phase gradient, these samples were analyzed and 33 metabolites of nine bioactive compounds were detected and tentatively identified by Metabolynx. Among the 33 metabolites, three metabolites were identified from plasma sample, three came from bile sample, and 27 metabolites were identified from urine and feces samples. This approach provided a rapid method for characterizing the metabolites of Dingzhi Xiaowan and gave the truly active structures and the action mechanism of their antidepressant effects.
Animals ; Bile ; metabolism ; Chromatography, High Pressure Liquid ; Depression ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; metabolism ; Feces ; chemistry ; Male ; Mass Spectrometry ; Medicine, Chinese Traditional ; Plant Extracts ; metabolism ; Rats ; Rats, Sprague-Dawley

Objective:To investigate the effect of DKK1 on linearly patterned programmed cell necrosis (LPPCN) and vasculogenic mim-icry (VM) and the related molecular mechanism in non-small cell lung cancer (NSCLC). Methods:A total of 173 human NSCLC speci-mens were collected to detect LPPCN by H&E staining, detect VM with CD31/PAS double staining, and investigate DKK1 and related protein expression by immunohistochemistry. The clinical pathological significance of LPPCN, VM, and DKK1 and the correlation of them were analyzed. Human NSCLC H460-DKK1 cells were engrafed in nude mice to evaluate the influence of DKK1 up-regulation on VM and LPPCN in vivo. Results:Approximately, 14.45%(25/173) of NSCLC had VM and 49.71%(86/173) had LPPCN. 25.6%(22/86) of NSCLC cases in LPPCN-positive group formed VM. Both of VM and LPPCN were all correlated with poor differentiation, late TNM stage, easy recurrence and metastasis and poor prognosis in NSCLC. DKK1 expression in the VM-positive group and the LPPCN-positive group was higher than that in the VM-negative group and the LPPCN-negative group, respectively. DKK1, LPPCN, and VM were positive-ly correlated with VE-cadherin, MMP-2,β-catenin nuclear expression and Twist1. H460-DKK1 transplantation tumor model confirmed that DKK1 promotes the expression of VM and LPPCN and related proteins in NSCLC. Conclusion:The increase of theβ-catenin and Twist1 expression induced by DKK1 may promote the formation of LPPCN and VM in NSCLC.