This study purposed to investigate the effects of different oxygen concentrations and reactive oxygen species (ROS) on the biological characteristics of hematopoietic stem cells (HSC) and their possible mechanisms through simulating oxygen environment to which the peripheral blood HSC are subjected in peripheral blood HSCT. The proliferation ability, cell cycle, directed differentiation ability, ROS level and hematopoietic reconstitution ability of Lin(-)c-kit(+)Sca-1(+) BMHSC were detected by using in vitro amplification test, directional differentiation test, cell cycle analysis, ROS assay and transplantation of Lin(-)c-kit(+)Sca-1(+) HSC from sublethally irradiated mice respectively. The results showed that oxygen concentrations lower than normal oxygen concentration, especially in hypoxic oxygen environment, could reduce ROS generation and amplify more primitive CD34(+)AC133(+) HSC and active CD34(+) HSC, and maintain more stem cells in the G(0)/G(1) phase, which is more helpful to the growth of CFU-S and viability of mice. At the same time, BMHSC exposed to normal oxygen level or inconstant and greatly changed oxygen concentrations could produce a high level of ROS, and the above-mentioned features and functional indicators are relatively low. It is concluded that ROS levels of HSC in BMHSCT are closely related with the oxygen concentration surrounding the cells and its stability. Low oxygen concentration and antioxidant intervention are helpful to transplantation of BMHSC.
Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Female ; Hematopoietic Stem Cells ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Oxygen ; administration & dosage ; pharmacology ; Reactive Oxygen Species ; metabolism

BACKGROUND: Streptococcus pneumoniae is the most common human pathogen causing community-acquired pneumonia. There is little information on the recent antimicrobial susceptibility patterns of S. pneumoniae in Busan and Gyeongnam of Korea. The aim of this study was to investigate the distribution and antimicrobial resistance of S. pneumoniae at 4 university hospitals in Busan and Gyeongnam. METHODS: We collected and analyzed the antimicrobial susceptibility results of 850 S. pneumoniae strains isolated from regional 4 university hospitals during the last 2 years from July 2013 through June 2015. RESULTS: Among 850 S. pneumoniae strains, 635 strains were isolated from respiratory specimens, followed by blood (N=121), CSF (N=13), and others (N=81). Antimicrobial susceptibility rates to penicillin, cefotaxime and ceftriaxone were 79.4%, 76.6% and 83.6%, respectively. The resistant rates to erythromycin and clindamycin were 80.9% and 68.2%, respectively. The resistant rates to levofloxacin were 9.2%. There were some differences in resistant rates by age groups, years, and specimen types. CONCLUSION: We found the changes of antimicrobial resistance of S. pneumoniae during the last 2 years. It is necessary to monitor the antimicrobial susceptibility of S. pneumoniae regularly for empirical therapy and for early detection of the changes of resistance.
Busan* ; Cefotaxime ; Ceftriaxone ; Clindamycin ; Drug Resistance ; Erythromycin ; Hospitals, University* ; Humans ; Korea ; Levofloxacin ; Penicillins ; Pneumonia ; Streptococcus pneumoniae* ; Streptococcus*

Polymerase chain reaction (PCR) has been used as a substitute for conventional serological methods in order to provide blood or blood products free from contaminating viruses and recently attempts have focused to detect 2 or 3 viruses by a single multiplex PCR (M-PCR) reaction. We were able to detect human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV), hepatitis B virus (HBV) and human cytomegalovirus (HCMV) simultaneously by a single M-PCR. However detection by gel electrophoresis of the products from M-PCR suffers from drawbacks such as low sensitivity and product sizes. Here we report enhanced detection systems of M-PCR based on nucleic acid hybridization with arrays built on membrane. Membrane array was manufactured by spotting appropriate probe DNAs on nylon membrane. Single or multiplex PCR was performed and the PCR products were labeled with DIG and allowed to hybridize with the membrane array. Results indicate that nonspecific hybridization was not observed for membrane DNA array. Additionally, membrane array method could detect small amount of viruses that were not detectable by conventional gel electrophoresis. At least 25-fold, and in some cases more than 125-fold increases in sensitivity was obtained with DNA array method. Thus, the nucleic acid hybridization with membrane array could be applied for the detection of M-PCR of viruses in blood or blood products.
Cytomegalovirus ; DNA ; Electrophoresis ; Female ; Hepacivirus ; Hepatitis B virus ; HIV-1 ; Humans ; Membranes ; Metrorrhagia ; Multiplex Polymerase Chain Reaction* ; Nucleic Acid Hybridization* ; Nylons ; Oligonucleotide Array Sequence Analysis ; Polymerase Chain Reaction

Objective To introduce an optimized practical method of making and detecting pipettes for microinjection.Methods Transfer pipette was made from hard glass capillary. We softened the hard glass capillary by rotating it in a spirit-lamp flame,then moved out from the flame and quickly pulled it into two transfer pipettes.After broken by a grinding wheel,the tip of the pipette was fire-polished by quickly touching the flame to make a fine opening.A hard glass capillary (1.0 mm,ouside diametre)was pulled into two holding pipettes by pipette Puller.The pipette shoulder was broken at desired position with a grinding wheel,then the fine pipette tip opening was heated by a microforge and shrinked into a diameter -15 μm.Injection pipette could be made directly from a capillary with filament by Puller.The solution loaded injection pipette and holding pipette were assembled into the micromanipulator and could be checked before use.We transfered both pipettes into the zygotes media drop,touched the holding pipette with the tip of the injection pipette to make a "suitable"opening.Then we switched injection pipette to the mineral oil and applied injection pressure through the injector to check whether the solution could come out of the tip smoothly and at a proper speed.It could be further verified by pronucleus microinjection of zygotes.Results The results showed that the method introduced in this paper could produce suitable pipettes for zygote microinjection.In particular,the method of detecting the opening of the injection pipette was helpful for achieving high efficiency of zygote microinjection.Conclusion The method introduced here to make and detect pipettes for microinjection is very helpful for establishing a standard microinjection manipulation procedure and improving the efficiency of zygote microinjection.

Objective A simple,sensitive and rapid ultra high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)method was developed and validated for the determination of ertapenem in human plasma.Methods Using ertapenem-D4 as internal standard,the protein in plasma was precipitated with acetonitrile;chromatographic column:ACQUITY HSS T3(2.1 mm × 50.0 mm,1.8 μm);the mobile phase was 0.1％formic acid aqueous solution(containing 2 mmol·L-1 ammonium formate)-acetonitrile(0.1％formic acid),using a gradient elution;flow rate:0.4 mL·min-1,injection volume:1 μL,column temperature:45 ℃,the analysis time was 4.5 min,the scanning mode is positive ion selective reaction monitoring mode(SRM)with an electric spray ion source(ESI).The specificity,standard curve and lower limit of quantification,precision and recovery,matrix effect,dilution effect and stability were investigated.Results Ertapenem had a good linearity within 0.5-80.0μg·mL-1,and the standard curve was y=4.25 × 10-1x-2.64× 10-2(r2=0.999 0),the lower limit of quantification was 0.5 μg·mL-1,the relative standard deviation within and between batches is 1.39％-4.15％.The extraction recovery rate was 58.36％-64.57％,and the relative standard deviation of dilution effect was 3.30％,and the matrix effect was 99.71％-103.23％.The relative standard deviation of room temperature,repeated freeze-thaw,4 ℃,and long-term stability are all less than 10％.Conclusion The method is sensitive,rapid and specific,which is suitable for clinical monitoring of Ertapenem.

This paper reviewed the research progress in the differentiation and treatment of precancerous lesions of gastric cancer(PLGC)with traditional Chinese medicine(TCM)in the recent 10 years.PLGC refers to the intestinal metaplasia and dysplasia of gastric mucosa developed from chronic atrophic gastritis.As an intermediate stage between chronic atrophic gastritis and gastric cancer,PLGC progresses slowly.TCM has certain advantages in the treatment of PLGC.PLGC can be mainly differentiated as five TCM syndrome types:spleen-stomach weakness syndrome,damp-heat in the spleen and stomach syndrome,qi stagnation in the liver and stomach syndrome,stasis obstruction in the stomach collateral syndrome,and stomach-yin deficiency syndrome.The popularization of gastroscopy technology has promoted the development of microscopic syndrome differentiation of gastric diseases.The combination of microscopic syndrome differentiation and macroscopic syndrome differentiation provides more theoretical supports for the TCM syndrome differentiation and treatment of PLGC.The basic pathogenesis of PLGC is due to the weakness of spleen and stomach and the disordered qi movement in the spleen and stomach,complicated by the accumulation of pathological factors such as damp-heat,stasis-toxin,phlegm-damp,and then the stomach-yin will be consumed over time.An amount of clinical studies on the treatment of PLGC with Chinese medicine have been carried out in recent years.The Chinese medicine treatment of PLGC is mainly through benefiting qi and invigorating spleen,assisted by the methods of soothing liver and activating blood,resolving dampness and eliminating stasis,resolving stasis and removing toxins,and nourishing yin and benefiting stomach,so as to prevent the PLGC progressing to gastric cancer.There are few studies on acupuncture treatment of precancerous lesions of gastric cancer.Therefore,it is necessary to carry out more strict and standardized multi-center large-sample RCTs,and to further study the therapeutic mechanism of acupuncture and moxibustion for PLGC,in order to provide more high-level evidence for the treatment of PLGC with TCM therapy.

BACKGROUNDThin endometrium is associated with poor reproductive outcomes; estrogen treatment can increase endometrial thickness (EMT). The aim of this retrospective cohort study was to investigate the factors influencing the effectiveness of estrogen treatment and reproductive outcomes after the treatment in patients with thin endometrium.

METHODSRelevant clinical data of 101 patients with thin endometrium who had undergone estrogen treatment were collected. Possible factors influencing the effectiveness of treatment were analyzed retrospectively by logistic regression analysis. Eighty-seven infertile women without thin endometrium who had undergone assisted reproduction served as controls. The cases and controls were matched for age, assisted reproduction method, and number of embryos transferred. Reproductive outcomes of study and control groups were compared using Student's t-test and the Chi-square test.

RESULTSAt the end of estrogen treatment, EMT was ≥8 mm in 93/101 patients (92.1%). Effectiveness of treatment was significantly associated with maximal pretreatment EMT (P = 0.017) and treatment duration (P = 0.004). The outcomes of assisted reproduction were similar in patients whose treatment was successful in increasing EMT to ≥8 mm and the control group. The rate of clinical pregnancy in patients was associated with the number of good-quality embryos transferred in both fresh (P = 0.005) and frozen-thawed (P = 0.000) embryo transfer cycles.

CONCLUSIONSThinner EMT before estrogen treatment requires longer treatment duration and predicts poorer treatment outcomes. The effectiveness of treatment depends on the duration of estrogen administration. Assisted reproductive outcomes of patients whose treatment is successful (i.e., achieves an EMT ≥8 mm) are similar to those of controls. The quality of embryos transferred is an important predictor of assisted reproductive outcomes in patients treated successfully with exogenous estrogen.

Endometrium ; drug effects ; Estrogens ; therapeutic use ; Female ; Humans ; Infertility, Female ; drug therapy ; therapy ; Male ; Pregnancy ; Pregnancy Rate ; Retrospective Studies

Objective:To investigate the inhibitory effects of Danshen injection against ovarian cancer cell proliferation induced by the interaction between platelets and cancer cells. Method:The induction of platelets on SKOV3 growth in vitro and the inhibitory effect of Danshen injection at 12，24，and 48 g·L^-1 were observed by 3-（4，5-Dimethylthiazol-2-yl）-2，5-diphenyltetrazolium bromide （MTT） and cell colony formation assays. The content of transforming growth factor-β₁ （TGF-β₁） in the platelet-tumor cell interaction system and platelet supernatant and the effect of Danshen injection on TGF-β₁ secretion were detected by enzyme-linked immunosorbent assay （ELISA）. The influences of tumor cell culture supernatant on platelet aggregation and secretion and the inhibitory effect of Danshen injection were determined by microplate assay and ELISA. The effects of Danshen injection on platelet nuclear factor kappa B （NF-κB） signaling pathway were assayed by Western Blot. Result:Compared with the blank group， the platelet induction group exhibited significantly elevated absorbance at A₅₇₀ （P<0.01）， while the absorbance at A₅₇₀ in the platelet + Danshen injection group was significantly lower than that in the platelet induction group （P<0.01）. The comparison with the Danshen injection group revealed that the cell proliferation inhibitory rate in the platelet + Danshen injection group at the same dose was more significant （P<0.01）. The number of colonies in the platelet induction group was obviously increased in contrast to that in the blank group（P<0.05）， while the number of colonies in the platelet + Danshen injection group was significantly lower than that in the platelet induction group（P<0.05，P<0.01）. As demonstrated by comparison with the blank group， TGF-β₁ content in the supernatant of the platelet induction group rose remarkably（P<0.01）， whereas that in the platelet + Danshen injection group declined（P<0.01）. Compared with the Danshen injection （24 g·L^-1） group， the platelet + Danshen injection group displayed more obvious inhibition（P<0.01）. Compared with the blank group， Danshen injection significantly reduced the TGF-β₁ content in platelet supernatant（P<0.05，P<0.01）. There was no significant change in the content of TGF-β₁ in SKOV3 supernatant treated with Danshen injection. The platelet aggregation， thromboxane A₂（TXB₂）， and serotonin （5-HT） secretion in the SKOV3 cell supernatant induction group were significantly increased as compared with those in the blank group （P<0.01）， while such indexes in the cell supernatant induction + Danshen injection group were obviously decreased （P<0.01）. Compared with the Danshen injection （24 g·L^-1） group， the cell supernatant induction + Danshen injection group displayed more obvious inhibition at the same dose（P<0.01）. Compared with the blank group， the platelet induction group exhibited obviously up-regulated phosphorylated TGF-β-activated kinase-1 （TAK-1） and NF-κB， but down-regulated phosphorylated inhibitory protein of NF-κB （IκB）（P<0.01）， which however were significantly reversed in the platelet + Danshen injection group（P<0.01）. Conclusion:Danshen injection affect the proliferation of SKOV3 cells by inhibiting their interaction with platelets， which may be related to the inhibited secretion of TGF-β₁.

OBJECTIVE: To observe the effect of electroacupuncture (EA) preconditioning on the expressions of tyrosine kinase Lyn and spleen tyrosine kinase (Syk) in mast cells of subcutaneous loose connective tissue in the rats with urticaria and explore the potential biological mechanism of EA in the intervention of urticaria. METHODS: A total of 32 SD rats were randomized into a blank group, a model group, an EA group and a positive medication group, 8 rats in each one. Except of the blank group, the passive cutaneous anaphylaxis (PCA) was adopted to prepare the model of urticaria in the rats of the rest three groups. In the EA group, EA was applied to bilateral "Quchi" (LI 11), "Xuehai" (SP 10) and "Zusanli" (ST 36), with disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in current intensity, once daily, for 20 min each time, consecutively for 7 days. In the positive medication group, loratadine (1 mg•kg•d) was for intragastric administration, once daily, consecutively for 7 days. The samples were collected for index detection 30 min after PCA antigen challenge in the rats of each group. Spectrophotometer was adopted to determine the effusion quantity of Evans blue in the allergized site of skin. HE staining was used to observe the morphological changes in the allergized site of skin. Toluidine blue staining was provided to observe mast cell degranulation in subcutaneous loose connective tissue in the allergized site of skin. Immunohistochemistry was applied to determine the protein expressions of Lyn and Syk during degranulation of mast cells. RESULTS: In the rats of the odel group, the eipdermis of allergized site was thickening, cells were disorganized in hierarchy and inflammatory cells were infiltrated largely in the dermis. In the positive medication group and the EA group, the epidermis was getting thin, cell arrangement was clear and the inflammatory cell infiltration was obviously alleviated as compared with the model group. Compared with the blank group, the OD value of skin dye effusion quantity, the degranulation rate of mast cells and the positive expressions of Lyn and Syk were all increased in the model group (<0.01). Compared with the model group, the OD value of skin dye effusion quantity, the degranulation rate of mast cells and the positive expressions of Lyn and Syk were all reduced in the EA group and the positive medication group (<0.01). Compared with the positive medication group, the degranulation rate of mast cells was increased significantly in the EA group (<0.01). CONCLUSION Electroacupuncture at "Quchi" (LI 11), "Xuehai" (SP 10) and "Zusanli" (ST 36) reduces vascular permeability and gives play to the role of anti-allergy by the way of regulating and controlling the degranulation of mast cells in the rats with urticaria and the effect mechanism of electroacupuncture may be related to the inhibition of protein expressions of Lyn and Syk in mast cells.
Acupuncture Points ; Animals ; Connective Tissue ; metabolism ; Electroacupuncture ; Mast Cells ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Syk Kinase ; metabolism ; Urticaria ; therapy ; src-Family Kinases ; metabolism

OBJECTIVETo investigate the effects of oxygen concentration and reactive oxygen species (ROS) on the biological characteristics of hematopoietic stem cells (HSC) and to analyzed the relationship among the oxygen concentration, ROS and the biological characteristics of mouse HSC through simulation of oxygen environment experienced by PB HSC during transplantation.

METHODSThe detection of reactive oxygen species (ROS), in vitro amplification, directional differentiation (BFU-E, CFU-GM, CFU-Mix), homing of adhesion molecules (CXCR4, CD44, VLA4, VLA5, P-selectin), migration rate, CFU-S of NOD/SCID mice irradiated with sublethal dose were performed to study the effect of oxgen concentration and reactive oxygen species on the biological characteristics of mouse BM-HSC and the relationship among them.

RESULTSThe oxygen concentrations lower than normal oxygen concentration (especially hypoxic oxygen environment) could reduce ROS level and amplify more Lin(-) c-kit(+) Sca-1(+) BM HSC, which was more helpful to the growth of various colonies (BFU-E, CFU-GM, CFU-Mix) and to maintain the migratory ability of HSC, thus promoting CFU-S growth significantly after the transplantation of HSC in NOD/SCID mice irradiated by a sublethal dose. BM HSC exposed to oxygen environments of normal, inconstant oxygen level and strenuously thanging of oxygen concentration could result in higher level of ROS, at the same time, the above-mentioned features and functional indicators were relatively lower.

CONCLUSIONThe ROS levels of BM HSC in PB HSCT are closely related to the concentrations and stability of oxygen surrounding the cells. High oxygen concentration results in an high level of ROS, which is not helpful to maintain the biological characteristics of BM HSC. Before transplantation and in vitro amplification, the application of antioxidancs and constant oxygen level environments may be beneficial for transplantation of BMMSC.

Animals ; Cell Differentiation ; Culture Media ; chemistry ; Erythroid Precursor Cells ; cytology ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Hematopoietic Stem Cells ; cytology ; metabolism ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Oxygen ; chemistry ; Reactive Oxygen Species ; metabolism