Objective The fluorescence-activated cell sorting (FACS) technique is a method for the identification and isolation of different cell populations.At present,the special surface marker for limbal stem cells has been not found yet.This study aimed to investigate the application of FACS technique in the research of rabbit limbal stem cells.MethodsCorneal limbal tissue was obtained from New Zealand white rabbits and cultured using the explant culture method in SHEM.Side population cells (SP cells) and non-SP cells were sorted from cultured rabbit limbal epithelium cells by FACS at a excitation wavelength 350 nm,and acquistion length 450 nm (blue light) and 675 nm (red light).The SP cells and non-SP cells were identified by detecting the expression of ABCG2 and K3/K12.The colony-forming efficiency of SP cells and non-SP cells were evaluated by the observation of cellular vitality with trypan blue staining.The percentage of colony formation was calculated as the colony number in various group/200×100%.ResultsIn 48-72 hours after primary culture,limbal epithelial cells migrated from the cultured tissue mass to form the mambrane-like structure and achieved 70%-80% confluence.The cells showed round,polygon and flattened shape.The proportion of SP cells in cultured limbal epithelial cells was 0.22%±0.09% with a colony-forming efficiency of 5.52±0.45% in SP cells and 0.78%±0.73% in non-SP cells,with a statistically significant difference between the two populations (t＝2.17,P＜0.01).After verapamil,an inhibitor of the expression of the ABCG2 protein,was added into the medium,the proportion of SP cells in the cultured limbal epithelial cells declined to 0.04%±0.006%.The SP cells presented a positive immunoresponse for ABCG2 and absence of immunoresponse for K3/K12,but a contradictory staining result was found in non-SP cells.ConclusionFACS can be applied in the research of limbal stem cells.

Totally 40 male SD rats were randomly divided into sedentary group (G1, n =8), exercise group (G2, n =16) and exercise with drug group (G3, n =16). Animal models were established by treadmill training. At the last time for training, rats in the G2 and G3 were separately divided into control group (n =8) and exhausting exercise group (n =8). Rats in the G3 were orally injected with quercetin 50 mg/kg by intragastric administration everyday at 3 hours before training, and others in the G1 and G2 were fed with the same amount of saline by gavage. Hemoglobin (Hb) content, activities of serum lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined in exhausted rats. The results showed that quercetin could remarkably restrain the decrease of Hb content and the increase of activities of ALT, AST and LDH after exhausted exercise.

Objective To investigate effect of hypothermia on coagulation function in major trauma patients and assess value of thromboelastography (TEG) monitoring.Methods Twenty-two patients with major trauma admitted to the emergency intensive care unit between January 2010 and June 2011 were enrolled in the study.The venous blood of the patients was sampled for TEG determination at different temperatures (37,35 and 33 ℃) to analyze variation of the indices including coagulation reaction time (R),clot formation time (K),rate of clot formation (Angle),maximum amplitude (MA)and coagulation index (CI).The patients were divided into normal coagulation group and abnormal coagulation group based on the CI value at 37 ℃ to analyze effects of temperature on TEG indices in both groups and their differences between groups.Results (1) Among 22 patients,TEG indices including R and K trended upward (P ＜ 0.01),but Angle,MA and CI trended downward (P ＜ 0.01) with decline of the temperatures.(2) K and Angle values,indicators of fibrinogen function,were obviously inhibited (P ＜ 0.05) with the temperature decreasing from 37 ℃ to 35 ℃,but other TEG indices had no significant changes.Whereas,all TEG indices were significantly inhibited when the temperature was decreased from 35 ℃ to 33 ℃.(3) There were significant differences in variation of each TEG index inhibited by hypothermia (P ＜ 0.01).All TEG indices showed significant differerces in the pairwise comparison,except for the differences between R and K as well as between Angle and MA (P ＜0.01).(4) R and K were increased,but Angle,MA and CI were decreased in both groups,with decline of the temperatures.Moreover,all TEG indices in the abnormal group were worse than those in the normal group.Conclusions Hypothermia has significant effect on coagulation function of patients with major trauma.TEG,which may be measured at any temperature,is more accurate in reflection of patients' actual coagulation function and is helpful for choice of an appropriate temperature in the mild hypothermia therapy.

BACKGROUND:The kidney tissues easily affected exercise ischemia reperfusion, increased free radicals and inflammation, resulted in abnormal renal function after acute exercise. OBJECTIVE:To observe the influence of Blackcurrant Extract on the structure of kidney and expression of tumor necrosis factor-α and nuclear factor-κB at 24 hours after exhaustiveexercise. METHODS:A total of 30 male Wistar rats were randomly divided into three groups (n=10). Rats in the Blackcurrant Extract group were intragastricaly administered 0.44 g/kg Blackcurrant Extract. Rats in the quietness control group and 24-hour exhaustive exercise group were intragastricaly given an equal volume of distiled water for 6 consecutive weeks. Rats in the 24-hour exhaustive exercise group and Blackcurrant Extract group received no swimming motion until exhaustion fatigue after final intragastric administration. Twenty-four hours later, samples were obtained. Kidney tissue morphology and ultrastructure were observed by electron microscopy and light microscopy. Protein expression of tumor necrosis factor-α and nuclear factor-κB was detected using immunohistochemistry. Tumor necrosis factor-αmRNA and nuclear factor-κB mRNA expression was detected using RT-PCR. RESULTS AND CONCLUSION:Compared with the quietness control group, tumor necrosis factor-α protein and nuclear factor-κB protein expression in the kidney was higher in the 24-hour exhaustive exercise group, and tumor necrosis factor-α mRNA and nuclear factor-κB mRNA expression was significantly increased (P < 0.01). Compared with the 24-hour exhaustive exercise group, tumor necrosis factor-α protein and nuclear factor-κB protein expression was lower in the Blackcurrant Extract group, and nuclear factor-κB mRNA expression was significantly decreased (P < 0.05); tumor necrosis factor-α mRNA expression was significantly reduced (P < 0.01). Kidney in the 24-hour exhaustive exercise group showed obvious morphological changes and ultrastructural damage. The structure of the kidney in the Blackcurrant Extract group tended to be normal. Results suggested that Blackcurrant Extract can repair the kidney tissue injury, reduce the expression of inflammatory factors, and prevent inflammatory damage in the kidney at 24 hours after exhaustive exercise.

Objective To evaluate clinical application value of critical flicker frequency (CFF),psychometric hepatic encephalopathy score (PHES) and Stroop test in the diagnosis of covert hepatic encephalopathy (CHE).Methods A total of 110 patients with decompensated liver cirrhosis and 54 individuals without liver diseases were enrolled as control group.According to PHES＜-4 points as reference threshold for CHE,the threshold of CFF and time of Stroop test for CHE diagnosis was caculated.Positive results of at least two of PHES,CFF and Stroop tests was considered as the gold standard for CHE diagnosis,and then the value of these three methods in CHE diagnosis was evaluated.Student's t test and receiver operating characteristic curve (ROC) were used for statistical analysis.Results Among the 110 patients with liver cirrhosis,40 patients had no hepatic encephalopathy (HE0),52 patients had CHE,and 18 patients had grade 2 hepatic encephalopathy (HE2).The CFF value and total time of Stroop test of control group were (43.70±1.92) Hz and (201.17±20.65) s,respectively.The CFF value of HE0 group was (41.40 ± 1.85) Hz,which was higher than that of CHE group ((38.33 ± 2.32) Hz),and the difference was statistically significant (t=-7.116,P＜0.01).The total time of Stroop test of HE0 group was (197.91±26.68) s,which was shorter than that of CHE group ((253.24± 33.33) s),and the difference was statistically significant (t=8.936,P＜0.01).When PHES＜-4 points was considered as a reference threshold of CHE,the threshold of CFF for CHE diagnosis was 39 Hz,the sensitivity was 94.9％ and the specificity was 73.1％,the area under the curve (AUC) was 0.879.The threshold of the total time of Stroop test for CHE diagnosis was 233.80 s,the sensitivity was 83.3 ％ and the specificity was 71.1％,the AUC was 0.803.The completion time of the number connection test (NCT)-A,NCT-B and digit symbol test (DST),which were there of five subtests of PHES,of CHE group were (80.27±36.05) s,(124.18±55.96) s and (25.03±8.23) s,respectively,compared with those of HE0 patients ((56.68±18.82) s,(80.00±25.58) s and (34.68±8.75) s,respectively),the differences were statistically significant (t =3.691,4.108 and-4.780;all P＜0.01).Compared with the results of combined PHES and Stroop test in the diagnosis of HE0,CHE and HE2,the consistency rates of CFF＜39 Hz as threshold for diagnosis were 95.0％,61.5％ and 100.0％,respectively.Conclusions NCT-A,NCT-B and DST three subtests of PHES have higher efficiency in CHE diagnosis.CFF and Stroop test are also reliable screening methods for CHE,with advantage of objectivity and high specificity.

Objective To evaluate the effects of modified ultrafiltration on the expression of aquaporin 1 (AQP1) in cardiopulmonary bypass (CPB)-induced lung injury in dogs.Methods Eighteen healthy adult dogs of either sex,weighing 15-20 kg,were randomly divided into 3 groups (n =6 each):control group (group C),group CPB and modified ultrafiltration group (group MUF).The dogs were anesthetized with intraperitoneal 2.5％pentobarbital 25 mg/kg.Thoracotomy was performed in all the three groups and in addition lung injury was produced by CPB in CPB and MUF groups.In group MUF,modified ultrafiltration was performed at 10-15 min after termination of CPB.Arterial blood samples were collected before mechanical ventilation (T1),at end of CPB (T2),and at 1 h after termination of CPB (T3) to calculate respiration index (RI) and oxygenation index (OI).The lungs were removed for microscopic examination of pathologic changes in lung tissues under light microscope and for detection of AQP1 mRNA expression by real-time PCR.Results RI and OI were significantly higher and AQP1 mRNA expression was lower at T2 and T3 than at T1 in CPB and MUF groups (P ＜ 0.05).Compared with group C,RI was significantly increased and AQP1 mRNA expression was down-regulated at T2,3 in CPB and MUF groups,and OI at T2.3 in CPB group and at T2 in MUF group was decreased (P ＜ 0.05).Compared with group CBP,RI was significantly decreased,OI was increased and AQP1 mRNA expression was up-regulated at T3 in group MUF (P ＜ 0.05).Conclusion Modified ultrafiltration can reduce CPB-induced lung injury in dogs and upregulation of AQP1 may be involved in the mechanism.

Objective To observe the destructive and scavenging effect of ambroxol (AMB) on the biofilm (BF) of Pseudomonas aeruginosa (P.a).To evaluate the synergistically bactericidal effect of AMB along with levofloxacin (LFX) on BF of P.a.Methods The early model (cultured for 3 d) and mature model (cultured for 7 d) of P.a wild strain (PAO1) BF were established,in vitro,respectively.The models were randomly (random number) divided into control group,AMB group and AMB + LFX group.The concentrations of AMB were 256 μg/ml and 512 μg/ml,respectively.When the early BF model and mature BF model were made,different concentrations of AMB were added in AMB group and AMB + LFX (1μg/ml) was added in AMB + LFX group.The number of viable P.a on the BF carrier was counted with the continuous dilution method 24 h after AMB or/and LFX added.Then,the BF morphological changes on the carrier surface were observed by using scanning electron microscopy (SEM).Measured data were analyzed with single factor analysis of variance (One-Way ANOVA).Results Both in the early BF model and in the mature BF model,the SEM examination showed that the BF in AMB group was significantly reduced compared to the control group,and this reduction of BF was in dose-dependent manner.LFX 1 μg/ml could reduce the number of viable bacterial in BF in both early model and mature model (P ＜ 0.05).LFX with addition of different concentrations of AMB showed stronger bactericidal effect than LFX used alone identified by more significant reduction in the number of colonv within the BF (P ＜ 0.05).Furthermore,the LFX combined with 512 μg/ml AMB reduced more significant number of colony apparently than the LFX combined with 256 μg/ml AMB (P ＜ 0.05).Conclusions AMB can destroy the early BF or mature BF partly,and LFX alone can partly reduce the number of viable P.a within BF.When LFX combined with AMB,they exert a synergistically bactericidal effect.

Objective To evaluate the role of aquaporin 1(AQPI)expression in the cartiopulmonary bypass(CPB)-induced lung injury in dogs.Methods Twenty-four healthy dogs,weighing 15-20 kg,were randomly divided into 4 group(n =6 each):control group(group C),acetazolamide Ⅰ group(group A Ⅰ),acetazolamide Ⅱ group(group A Ⅱ),and acetazolarnide Ⅲ group(group AⅢ).Lung injury was produced by CPB.The traditional priming solution was infused in group C.Priming solutions containing acetaaolamide 20,40 and 60 mg/kgwere infused in groups A Ⅰ,A Ⅱ and A Ⅲ respectively.Blood samples were collected from the femoral artery before mechanical ventilation,at the end of CPB and at 1 h after end of CPB(T1-3)for arterial blood gas analysis.Respiration index(RI)and oxygenation index(OI)were calculated.The lung specimens were oblained for determination of AQPI mRNA and protein expression(by RT-PCR and Western blot)and for microscopic examination.The pathological changes of the lung were scored.Results Compared with group C,P(A-a)O2,RI and the pathological score were significantly increased at T2.3,OI was significantly decreased at T2.3,and AQP1 protein expression was down-regulated at T2.3 in groups A Ⅰ,A Ⅱ and AⅢ,and AQP1 mRNA expression was down-regulated at T2.3 in groups AⅡ and AⅢ(P＜0.05).Compared with group A Ⅰ,P(A-a)O2,RI and the pathological score were significantly increased at T2.3,OI was significantly decreased at T2.3,and AQP1 protein expression was down-regulated at T2.3 in groups A Ⅱ and AⅢ,and AQP1 mRNA expression was down-regulated at T2.3 ingroup A Ⅲ(P ＜ 0.05).Compared with group A Ⅱ,RI and the pathological score were significantly increased at T2.3,and AQP1 protein expression was down-regulated at T2.3 in group A Ⅲ(P ＜ 0.05).Conclutsion Down-regulation of AQPI expression is involved in the CPB-induced lung injury in dogs.

This study aimed at exploring the inhibitory effect behind its mechanism on acid-soluble polysaccharides from G.incamatum in transplanted H22 tumor mice.Different indices,including tumor inhibitory rate,organ index of liver,thymus and spleen,IL-2,IFN-γ and TNF-α were detected for the evaluation of anti-tumor effects and the mechanism.Furthermore,HE staining and TUNEL assay were adopted to investigate the pathological changes of tumor tissue and cell apoptosis,respectively.As a result,the three dose groups of acidsoluble polysaccharides of G.incamatum successfully inhibited the proliferation of tumor cells,while organ indexes of spleen and thymus were improved and serum IL-2,IFN-γ and TNF-α increased.H&E staining and TUNEL assay showed the polysaccharides induced cell apoptosis,playing a significant role in the inhibition of tumor growth.In conclusion,acid-soluble polysaccharides of G.incamatum possessed significant anti-tumor effects,behind which the mechanism could be related to the regulation of immune regulation,cell apoptosis,and the protection of liver function.

Objective To compare the influences of different dilution titers on the ANA detection by the indirect immunofluores‐cence assay(IIF) in children for investigating the necessity of reducing serum initial dilution titer .Methods Serum ANA was detec‐ted by using the indirect immunofluorescence assay at a serial of dilution titer in 110 healthy controls and the results were compared with the results of specific ANAs by the linear immunoassay (LIA);meanwhile the ANA‐LIA results in clinical children patients with ANA‐IIF negative were also analyzed .Results With the dilution titers gradual decrease from 1∶80 ,1∶40 and 1∶20 in the samples of the health group ,the positive detection rates of ANA‐IIF were risen ,which were 7 .3% ,9 .1% and 10 .9% respectively , but the differences were not statistically significant (P>0 .05) ,the weak‐positive rates were 7 .3% ,15 .5% and 31 .8% respective‐ly ,the differences were statistically significant (P<0 .01) .Among 110 healthy children under going the physical examination ,the specific ANA was detected out in 8 samples ,the positive rate was 7 .3% .Among 8 positive cases at the dilution titer of 1∶80 by the IIF method ,specific ANA was in 2 cases;in 4 added cases of fluorescence ANA positive samples at the dilution titers of 1∶40 and 1∶20 ,specific ANA was in 1 case .If with any positive of ANA‐IIF(1∶80) or ANA‐LIA as the ANA positive ,the ANA positive rate was risen from 7 .3% to 12 .7% .In the clinical samples among 29 cases of ANA‐IIF(1∶80) negative autoimmune liver disease related autoantibody detection ,the specific ANA‐LIA positive was detected in 5 cases (17 .2% ) .Conclusion Reducing the initial ti‐ter of children serum is unable to obviously increase the ANA‐IIF positive detection rate ,on the contrary increases the non‐specific weak positive .Therefore ,clinical laboratory does not change the dilution titer of children routine ANA sample .The detection by combining with the specific ANA‐LIA spectrum is conducive to find ANA .