BACKGROUND:Separating umbilical cord blood stem cells using tubes has low efficiency,and microbial contamination easily occurs during this process,therefore,safety cannot be ensured in clinical application.lt is urgent to find a method for separating umbilical cord blood stem cells to treat femoral head necrosis.OBJECTIVE:To establish a high efficient,safe,and clinically valuable method to separate umbilical cord blood stem cells.DESIGN,TIME AND SETTING:A self-control experiment was performed at the First Department of Surgery,Zhengzhou Second People's Hospital,Institute of Blood Constituent Application,Henan Red Cross Blood Centre between February 2006 and August 2007.PARTICIPANTS:Eight male patients with femoral head necrosis,averaging 40.6 years of age,were included in this study.Of these patients,4 had the history of hormone application.An average of 90 mL umbilical cord blood was harvested from each healthy normal full term neonate from Maternal and Children Health Care Hospital of Zhengzhou City.The quadruple bags used for separating umbilical cord blood stem cells consisted of 1 main bag,1 empty bag,and 2 physiological saline bags,provided by Shandong Weigao Holding,China.METHODS:Within 6 hours after collection,umbilical cord blood was centrifuged in the empty bag of quadruple bag,which was connected with an aseptic filling machine.After centrifugation,partial blood plasma was discarded,and the remaining erythrocytes were thoroughly mixed by adding hetastarch.Five minutes later,the mixture was diluted with physical saline at 1:1.Umbilical cord blood was slowly added into the main bag (at 1:1),in which,human lymphocyte separating medium was pre-added.After cantrifugation,the upper layer of solution,i.e.,monocyte-rich solution,was transferred into another empty bag.Within 24hours of preservation,after suspension with umbilical blood plasma,umbilical cord monocytes were transfused into patients with femoral head necrosis via superficial vein on the hand back,monocytes≥1×108/portion,2 portions once.There were three treatment courses,each involving three transfusion sessions,one session every 4 days,and a 2-3-month interval between two treatment courses.MAIN OUTCOME MEASURES:Cell recovery rate and cell viability of umbilical cord blood monocytes and improvements in clinical symptoms.RESULTS:The separation of quadruple bags could obtain umbilical cord blood monocytes with high recovery rate.Furthermore,microbial contamination hardly occurred in the process of separation.Hip joint pain relieved or disappeared to different extents in all 8 patients,with an effective rate of 100%.Abduction and internal rotation of hip joint,ambulation distance,and gait were markedly improved.At 6 months after cell transplantation,5 patients presented with changed bone density in femoral head necrosis regions,2 showed normal femoral head morphology,and the remaining 1 exhibited no obvious changes.Joint effusion was reduced or disappeared in 12 hips.Magnetic resonance images showed that femoral head morphology had been improved in various degrees in 9 hips,but no changes in 3 hips.No complications,fever,or allergies occurred during and after cell transplantation.CONCLUSION:The method of separating stem cells from umbilical cord blood in junction with aseptic interface technology is highly effective,safe,and clinically valuable.Multiple intravenous transfusions of umbilical cord blood stem cells provide a novel approach for systemic treatment of femoral head necrosis.

Background Proliferation of the human Tenon capsule fibroblasts(HTFs) is a main cause of failure of filtering surgery.To search the drug of inhibiting the growth of the HTFs is essential for the improvement of successful rate of filtering surgery.Objective The aim of this study was to investigate the inhibitory effect of apigenin on HTFs and its mechanism.Methods Human Tenon capsular tissue was obtained during the strabismus correction surgery.HTFs was primarily cultured using explant method and identified using vimentin by immunochemistry.The 3-5 generation of cells were incubated to 96-well plate.Apigenin of 0,20,40,80,160 μmol/L was added into the medium,respectively,for 24,48,72 hours,and the proliferation of HTFs was detected by sulfonyl chloride (SRB) at the wavelength of 560 nm (A560).Bromodeoxyuridine (BrdU) of 10 μg/L was added to culture the cells for 48 hours to calculate the labeling rate of BrdU.The morphology of the cells was observed using Hoechst 33258 staining,and apoptosis and cells cycle were evaluated by flow cytometry.Results Cultured cells grew well with the positive response for vimentin,showing the green fluorescence in cytoplasm.SRB assay showed that the A560 value was gradually declined with the increase of the dosage of apigenin and prolong of time (Fgroup =480.306,P =0.000 ; Ftime =555.144,P =0.000).The labeling rate after 0,40,80 μmol/L apigenin acted for 48 hours was (87.860 ±0.632)％,(61.520±4.306)％ and (23.480±4.472)％,showing a significant difference among the three groups (F =299.347,P =0.000).The labeling rate of HTFs for BrdU was significantly decreased in the 40 and 80 μmol/L apigenin groups compared with the 0 μmol/L apigenin group (P＜0.05).Hoechse 33258 staining found that the number of the HTFs was gradually decreased and the cell number of karyopyknosis and nuclear deformation was increased with the increase of apigenin dosage.Percentage of cells in G0/G1 phase were raised and that in S and G2/M phase were declined in the higher dosage apigenin group,with a significant difference among the different groups (FG0/G1 =58.621,P=0.000;Fs =32.357,P=0.001 ;FG2/M =83.998,P=0.000).In the 72nd hour after acted by 0,40,80,160 μmol/L apigenin,the apoptosis rate of HTFs was (4.77±0.21) ％,(13.24±1.35)％,(18.33±1.86) ％,(31.58 ± 2.77) ％,respectively,with a statistically significant difference among the four groups (F =204.791,P＜0.05).Conclusions Apigenin restrains the growth of HTFs by evoking G0/G1 cell cycle arrest and inducing apoptosis in a dosage-and time-dependent manner.

Objective To study the effects of oxymatrine as inhibitor of hemorrhagic fever with renal syndrome virus (HFRSV) infection in vitro and in vivo.Methods In vitro studies,a dose of oxymatrine without cytotoxicity and 76-118 strain of HFRSV was taken to treat Vero cells in three ways:①After treated with oxymatrine for 48 h,Vero cells were attacked by HFRSV at dilution of 10-1 ～ 10-6,respectively for 24 h before changing to maintenance medium; ②Vero cells were first attacked by HFRSV of 10-1 ～ 10-6 dilution respectively,then oxymatrine was used for 48 h before changing to maintenance medium; ③Vero cells were attacked by HFRSV at dilution of 10-1 ～ 10-6 respectively,and meanwhile treated with oxymatrine for 48 h before changing to maintenance mcdium.Each dilution handled four porocytes,and four positive controls were set up at the same time.Indirect immunofluorescence assay (IFA) was performed to determine the inhibitory effect of oxymatrine in experimental group and positive control.In vivo studies,thirty 2-week-old hamsters,weighing about 30-40 g,were divided into experimental and control groups according to body weight,n =15.These aninals were inoculated intraperitoneally with HFRSV in 100TCID50(0.1 ml each); on days 4-13,0.1 ml of oxymatrine 1:100 were given to each hamster in experimental group daily by intraperitoneal injection,while the same amount of saline was given to the control ones.Lung tissue of hamsters was then dissected out to slice to be identified by immunofluorcscence stain.Results It was demonstrated that oxymatrine with the diluted fractions of 1:8 was safe in vitro.When the virus dilution of HFRSV was l0-4,compared with control groups,the differences were statistically significant in method 2 and 3 (z =-2.53,-2.53,all P ＜ 0.05),while no statistical significance in method 1 (z＝5.36,P＞ 0.05).When the virus dilution of HFRSV was 10-1 ～ 10-3,10-5,10-6,the differences were not statistically significant (z--0.00,-0.32,-0.19,4.21,4.21,all P ＞ 0.05).In vivo studies,compared with control group,the differences were statistically significant in experimental group (z =-3.85,P ＜ 0.05).Conclusion Oxymatrine significantly inhibites HFRSV.

Animals ; Disease Models, Animal ; Liver ; pathology ; Liver Failure, Acute ; metabolism ; pathology ; Male ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Transcription Factor RelA ; metabolism

Objective To explore the chronic effects of mild and moderate iodine excess and iodine restriction on apoptosis of thyrocytes. Methods Wistar rats were exposed to 4 different doses of iodine: 4 μg/d (control), 6 μg/d (1.5 fold iodine excess), 12 μg/d (3 fold iodine excess), and 24 μg/d (6 fold iodine excess) for 1, 2, 4 and 8 months. Some rats treated for 8 months were fed with 4 μg/d iodine for another 3 months. Urinary iodine concentration was monitored by arscnic/cerium catalyzing spectrophotography. Apoptosis was determined by flow cytometry after Annexin V-FTTC staining and uhrastructure assessment under electronic microscope. Cell cycle kinetics was analyzed by flow eytometry after propidium iodine staining. Fluorescent measurement by DCFH-DA probe was used to determine the intracellular reactive oxygen species (ROS) level. Expressions of apoptic proteins were analyzed by flow cytometry and immunohistochemistry. Results Apoptotosis rate and ROS production in thyrocytes were significantly increased in 3 and 6 fold iodine excess groups after 4 months and 8 months (all P < 0.05), which was reversed with iodine restriction. 6 fold iodine exposure was proved to cause a reduction of cells in GOG1-phase (64% and 67% vs 80%, both P < 0. 05) and a concomitant accumulation in S-phase (5% and 6% vs 3%, both P <0.05) after 4 months and 8 months. Expressions of Fas, FasL and TRAIL proteins in 3 and 6 fold iodine excess groups after 8 months were increased by 2 to 4 times compared with control group and did not return to normal after iodine restriction. Bcl-2 and Bax remained constant. Positive correlations were observed among iodine amount, apoptosis rate and ROS level in 6 fold iodine excess group after 8 months (r = 0. 637-0.790, P < 0.01). Conclusion Chronic iodine excess results in thyrocyte apoptosis due probably to generation of ROS.

OBJECTIVEApoptosis of the cells of liver cancer cell line HepG2 could be induced by TNF alpha and actinomycin D (Act D). In the current study, the molecular mechanism of the apoptosis protection of stronger neo-minophagen C (SNMC) to HepG2 cells was investigated.

METHODSSNMC was added to the HepG2 cell culture medium when the cell concentration reached 0, 2, 20, 100, 200, 800 microg/ml 30 min before their apoptosis were inducted with TNF alpha and Act D. A flow cytometry assay was performed to detect the cell apoptosis rate; electromicroscopy was employed to visualize the subcellular structure after apoptosis. DNA ladder formation was checked with genomic DNA agarose electrophoresis. The expression pattern of apoptosis related protein Caspase-3, Bcl-2 and Bax was detected by Western blot.

RESULTSAfter pretreatment with various concentrations of SNMC and 12 hours after treatment with TNF alpha and Act D, the HepG2 cell apoptosis rate and DNA ladder formation decreased dramatically when the SNMC concentration was higher in the media; the intracellular inactive form of Caspase-3 increased while the 17*10(3) active Caspase-3 decreased gradually. In addition, the expression of Bcl-2 increased and the expression of Bax decreased. Under the electromicroscope, the typical nucleolus condensation of HepG2 induced by TNF alpha and Act D was not seen among the 100 microg/ml SNMC treated cells.

CONCLUSIONSNMC inhibits TNF alpha and Act D induced HepG2 cell apoptosis. This protective action may be regulated by intracellular apoptosis related factors.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cysteine ; pharmacology ; Drug Combinations ; Glycine ; pharmacology ; Glycyrrhiza ; Humans ; Liver Neoplasms ; pathology ; Oleanolic Acid ; analogs & derivatives ; pharmacology

BACKGROUND: Benzalkonium chloride has been used in dental restorative materials to enhance the long-lasting antibacterial properties of materials. OBJECTIVE: To evaluate the antibacterial activity of benzalkonium chloride on oral common pathogenic bacteria in vitro. METHODS: The agar diffusion method was used to determine the inhibitory effects of antibacterial agents, 0.1% benzalkonium chloride, 3% H2O2and 5.25% sodium hypochlorite, on five kind of oral pathogens, Porphyromonas gingivalis (P.g), Prevotella intermedia (P.i), Actinobacillus actionmycemcomitans (A.a), Streptococcus mutans (S.m) and Enterococcus faecalis (E.f). The tube dilution method was used to detect the minimal inhibitory concentration of benzalkonium chloride against the five bacteria mentioned above. RESULTS AND CONCLUSION: The antibacterial effect of 0.1% benzalkonium chloride on P.g was not significantly different from that of 3% hydrogen peroxide (P＞0.05), while 0.1% benzalkonium chloride showed better effect on P.i than 3% hydrogen peroxide (P＜0.05). On P.g and P.i, the antibacterial effect of 0.1% benzalkonium chloride was worse than that of 5.25% sodium hypochloritethe ( P＜0.05). The antibacterial effect of 0.1% benzalkonium chloride on A.a and S.m was better than that of 3% hydrogen peroxide (P＜0.05), and similar to that of 5.25% sodium hypochlorite (P＞0.05). The antibacterial effect of 0.1% benzalkonium chloride on E.f was better than that of 3% hydrogen peroxide (P ＜ 0.05), but worse than that of 5.25% sodium hypochlorite (P ＜ 0.05). The minimal inhibitory concentration of benzalkonium chloride to P.g, P.i, A.a, S.m, E.f was 16, 2, 4, 2, 4 mg/L, respectively. To conclude, 0.1% benzalkonium chloride has strong antibacterial effects on P.g, P.i, A.a, S.m and E.f.

Objective To explore the effect of multi-disciplinary team (MDT) combined with bundle management on prevention and control of multidrug-resistant organism (MDRO) infection in the intensive care unit(ICU).Methods Patients who were admitted to the ICU in a tertiary first-class hospital from January 2013 to December 2015 were studied,MDT combined with bundle management has been applied in the prevention and control of MDRO infection in ICU since January 2014,continuous quality improvement program was performed one year later,isolation of MDROs from specimens of ICU patients before implementation(in the year of 2013),after implementation(in the year of 2014),and after continuous quality improvement(in the year of 2015) was compared.Results The infection rates of MDROs in ICU patients before implementation,after implementation,and after continuous quality improvement were 26.55％ (154/580),17.13％ (117/683),and 12.01％ (77/641) respectively,showing a downward trend,with a significant difference (x2 =44.030,P＜0.001);the total isolation rates of MDROs in ICU patients were 64.44％(154/239),63.59％(117/184),and 43.26％ (77/178) respectively,showing a downward trend,with a significant difference (x2 =22.284,P＜0.001).The main MDROs in ICU were multidrug-resistant (MDR) and pandrug resistant(PDR) Acinetobacterbaumannii (44.54％).Conclusion MDT combined with bundle management can decrease MDRO infection rate and isolation rate in ICU.

OBJECTIVETo investigate the protective effect of stronger neo-minophagen C (SNMC) on fulminant liver failure (FLF).

METHODSD-Gal N and LPS were injected once into the abdominal cavity of rats to establish an experimental model of FLF. The level of plasma ALT, Alb, TBil, TNFalpha, NO, ET-1, IL-6 and liver histopathology of the rats were examined.

RESULTSIn the D-Gal N and LPS model of FLF, there was an obvious decline of plasma TNFalpha (F = 52.84), NO (F = 15.81), ET-1 (F = 15.68), IL-6 (F = 15.32) and there was less hepatic tissue damage in SNMC-treated groups using different doses (high dose, medium dose, low dose) and at different times (pre-protection, simultaneous protection, post-protection) compared with those not treated with SNMC. These results indicated that SNMC could be used to treat FLF. It was better to use a low dose of SNMC and use it at the same time as inducing the FLF. There were no differences in the results of those treated with SNMC of different dosages and treated at different times.

CONCLUSIONSNMC can decrease the mortality of FLF by preventing hepatocyte apoptosis induced by D-Gal N and LPS and inhibit liver inflammation caused by all kinds of factors.

Animals ; Anti-Inflammatory Agents, Non-Steroidal ; therapeutic use ; Female ; Galactosamine ; Glycyrrhizic Acid ; therapeutic use ; Lipopolysaccharides ; Liver Failure, Acute ; chemically induced ; drug therapy ; Male ; Mice

OBJECTIVETo investigate the effect of rhizoma paridis total saponins(RPTS)on the levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) in blood serum of two-hit rat model induced by multiple fractures and lipopolysaccharide.

METHODSSixty-eight Wistar rats were randomly divided into five groups. The models were made in four groups (expect the blank control group) in accordance with the standard of two-hit animal model induced by multiple fractures and lipopolysaccharide. At 1 hour after models made,the rats in RPTS groups were given rhizoma paridis total saponins with different concentrations by intragastric administration. Six hours later, the concentrations of TNF-alpha, IL-1 beta and IL-6 in the blood serum of all rats were detected by ELISA (enzyme linked immunosorbent assay).

RESULTSThe concentrations of TNF-alpha, IL-1 beta and IL-6 in blood serum of rats in the model group were remarkably higher than those in the blank control group (P<0.001), and these in the RPTS groups were remarkably lower than those in the model group (P<0001).

CONCLUSIONRPTS can decrease the levels of TNF-alpha and IL-6 and IL-1 beta in the blood serum of rats subjected to two-hit induced by multiple fractures and lipopolysaccharide.

Animals ; Cytokines ; blood ; Female ; Fractures, Bone ; blood ; drug therapy ; metabolism ; Lipopolysaccharides ; metabolism ; Male ; Multiple Trauma ; blood ; drug therapy ; metabolism ; Rats ; Rats, Wistar ; Rhizome ; chemistry ; Saponins ; pharmacology ; therapeutic use