Objective To explore the application of PCR in detection and diagnosis of Echinococcus granulosus and develop a diagnostic technique by DNA hybridization labeled with DIG.Methods A pair of primers were designed according to Echinococcus granulosus gene fragment sequence, and the special primers were as: P1 5′-GGAATGGAGAGAAGTTAC-3′ P2 5′-GCAACCTCCGGAACTTGC-3′. The hydatid fluid, secondary hydatid and protoscolex of Echinococcus granulosus were used as template, 417 bp special band was got after being amplified by PCR. The PCR product was purified and labeled with DIG. The special DNA probe was successfully got and was used to detect the Echinococcus granulosus. Results The DNA from Escherichia coli, Shigella, Tubercle, Cysticercus granulosus and healthy human leukocyte were extended by PCR and hybrided by DNA probe labeled with DIG, and only Cysticercus granulosus expressed 471 bp special band. The sensitivity of PCR was that one Cysticercus granulosus can be detected or 100-10 fg DNA, while the dot hybridization was 2500 fg. Heterologous DNA did not be positive reaction, even increasing the spotting membrane dosages. Conclusion The technique of PCR labeled with DIG showed good specificity, high sensitivity, more accuracy and quickness and so it can provide scientific basis for early diagnosis and epidemiological investigation of Echinococcus granulosusEXPERIMENTAL RESEARCH WITH PCR AND DNA HYBRIDIZATION TECHNIQUE IN DETECTION AND DIAGNOSIS OF ECHINOCOCCUS GRANULOSUS$$$$ Feng Xiaomei, Ji Hugang, Cao Yinfang, Wang Wenhao (Department of Clinical Laboratory, Inner Mongolia Autonomous Region Hospital, Huhhot 010017, China) Abstract Objective To explore the application of PCR in detection and diagnosis of Echinococcus granulosus and develop a diagnostic technique by DNA hybridization labeled with DIG.Methods A pair of primers were designed according to Echinococcus granulosus gene fragment sequence, and the special primers were as: P1 5′-GGAATGGAGAGAAGTTAC-3′ P2 5′-GCAACCTCCGGAACTTGC-3′. The hydatid fluid, secondary hydatid and protoscolex of Echinococcus granulosus were used as template, 417 bp special band was got after being amplified by PCR. The PCR product was purified and labeled with DIG. The special DNA probe was successfully got and was used to detect the Echinococcus granulosus. Results The DNA from Escherichia coli, Shigella, Tubercle, Cysticercus granulosus and healthy human leukocyte were extended by PCR and hybrided by DNA probe labeled with DIG, and only Cysticercus granulosus expressed 471 bp special band. The sensitivity of PCR was that one Cysticercus granulosus can be detected or 100-10 fg DNA, while the dot hybridization was 2500 fg. Heterologous DNA did not be positive reaction, even increasing the spotting membrane dosages. Conclusion The technique of PCR labeled with DIG showed good specificity, high sensitivity, more accuracy and quickness and so it can provide scientific basis for early diagnosis and epidemiological investigation of Echinococcus granulosus[infections.

Objective To investigate the effects of Triptolide on apoptosis of cultured rat mesangial cells treated by TGF-β1 and the role of Smac in this process. Methods The mesangial cells were pre-treated with different concentrations of Triptolide for 24 hours, then stimulated with TGF-β1 for 24 hours. Apoptotic cells were detected by TUNEL assay. Smac transcription level was determined by Real time-PCR analyses. Smac expression level was assessed using Western blot anal-yses. Localization of Smac was shown by confocal fluorescence microscopy. Results Compared with control group, TGF-β1 inhibited apoptosis and Smac transcription and expression in rat mesangial cells. By contrast, Triptolide promoted mesangial cells apoptosis. In Triptolide groups, Smac mRNA and protein levels were up-regulated. Additionally, in normal and TGF-β1 groups Smac protein was mainly localized in mitochondriawhile in Triptolide groupit was mainly localized in cytoplasm and nucleus with increased fluorescence intensity. Conclusion Triptolide could promote the effect that TGF-β1 inhibited apop-tosis of mesangial cells, through both up-regulation the expression of Smac and stimulating it translocation from mitochon-dria to cytoplasm and nucleus.

Objective To understand the experiences and suggestions for clinical practice training of emergency nurses in suburb of Shanghai City,and to provide theoretical basis for further improving clinical practice training of nurses in emergency department.Methods Using the phenomenological methods and Colaizzi 7 step analysis method to refine the theme,11 interviews were conducted.Results Respondents believed emergency nurses in clinical practice training was important; Training content should follow the training outline and needed appropriate extension according with the demand of the regional students,and help to improve the students' practical ability to work.The students hoped to gain more knowledge.PBL teaching method and situational simulation assessment mode were used.The teachers' solid professional knowledge,skills,and teaching ability and professional dedication were strong.Conclusions We should listen to the voices of training students,adopt relevant suggestions,and gradually improve the clinical practice training of emergency nurses mode.

Objective To explore the relationship between serum HBV DNA level , expression of PD-1 on CTL and liver functions in patients with hepatitis B virus cirrhosis. Methods 109 HBV hepatic cirrhosis patients with , HBV DNA positive , HBeAg positive and HLA-A2 positive were divided into two groups according to their HBV DNA levels,52 cases in group A, HBV DNA 2-4 log10 copies/mL, 57cases in group B, HBV DNA 5-7 log10 copies/mL , differences of HBV specific CTL surface PD-1 expressions , HBV specific CTL levels and liver functions were compared between two groups. Results HBV specific CTL surface PD-1 expressions of group A were lower than that of group B (t = 11.101, P < 0.01), HBV specific CTL levels of group A were higher than that of group B (t = 24.424, P < 0.01), ALT of group A were lower than that of group B (t = 2.652, P < 0.01), ALB of group A were higher than that of group B (t = 2.347, P < 0.05). Child-pugh rating, grade C of group A was lower than that of group B (χ2= 4.262,P < 0.05). Conclusions HBV specific CTL surface PD-1 expression levels were higher in those hepatic cirrhosis patients with , high level of serum HBV DNA, and HBV specific CTL levels were lower, liver functions damage were much serious. So, lower HBV DNA levels , lower HBV specific CTL surface PD-1 expression levels and higher HBV specific CTL levels can product relatively good effect of anti-viral treatment.

Objective To explore the changes of programmed death receptor-1 (PD-1) in chronic hepatitis B (CHB) patients with different baseline of hepatitis B virus (HBV) DNA levels after treatment with adefovir dipivoxil (ADV).Methods One hundred CHB patients with positive hepatitis B e antigen (HBeAg),1 × 104 copy/mL≤HBV DNA≤1 × 107 copy/mL,and positive human leukocyte antigen-A2 were divided into two groups according to the baseline HBV DNA level:47 cases in low virus load group whose HBV DNA level was ≤1 × 105 copy/mL; 53 cases in high virus load group whose HBV DNA level was＞1 × 105 copy/mL.Both groups were treated with ADV 10 mg/d.Serum HBV DNA,HBeAg seroconversion rate,alanine aminotransferase (ALT) and total bilirubin (TBil) levels of both groups before treatment and 12 months after treatment were compared.Flow cytometry was used to test peripheral blood HBV-specific cytotoxic T lymphocyte (CTL) surface PD-1 and peripheral blood HBV-specific CTL level.Categorical data were tested by x2 test; quantitative data was compared with t-test.Results Peripheral blood HBV-specific CTL surface PD-1 of CHB patients in low virus load group was 20.17 ％±1.69％,which was lower than that in high virus load group (41.38％±2.30％,t =53.02,P＜0.01) ; peripheral blood HBV specific CTL levels in two groups were 0.37％±0.02％ and 0.17％± 0.02％,respectively (t=50.47,P＜0.01) ; ALT and TBil levels in low virus load group were both lower than those of high virus load group (t=13.07,P＜0.01; t=5.06,P＜0.01).Twelve months after treatment,HBV DNA of 25 cases (53.2％) in low virus load group and 10 cases (18.9％) in high virus load group were lower than the detectable level (HBV DNA＜500 copy/mL,x2 =12.89,P＜0.01);HBeAg seroconversion was achieved in 15 cases(31.9％) and 1 case (1.9％),respectively (x2 =16.72,P＜0.01) ; peripheral blood HBV-specific CTL surface PD-1 expression levels were 9.00 ％ ±1.38 ％ and 29.40 ％ ± 3.76 ％,respectively (t =36.80,P＜ 0.01) ; peripheral blood HBV-specific CTL levels were 0.65％±0.10％ and0.48％±0.07％,respectively (t=9.61,P＜0.01).Conclusions After treatment with ADV,along with the decrease of HBV DNA load,HBV-specific CTL surface PD-1 expression decreases,while HBV-specific CTL level increases.The changes in low virus load group are much more remarkable.

Objective To investigate the association between the difference of specific cytotoxic lymphocyte (CTL) and liver function of patients with hepatitis B virus (HBV) genotype B and C infections and interleukin (IL)‐7 induced follicular helper T lymphocytes (Tfh) .Methods Sixty‐seven patients with chronic hepatitis B (CHB) hospitalized at Wuxi No .5 People′s Hospital from August 2013 to January 2015 were collected and 30 healthy blood donors were set as healthy control group .The peripheral blood IL‐7 , Tfh ,IL‐21 ,HBV specific‐CTL ,nonspecific CTL ,levels of HBV DNA ,alanine aminotransferase (ALT) and total bilirubin (TBil) were compared between patients with genotype B and C infection ,hepatitis B e antigen (HBeAg)‐positive and HBeAg‐negative CHB ,high ALT level and low ALT level .Multivariate regression analysis was performed to determine the factors associated with IL‐7 .The t test was used for quantitative data and chi‐square test was used for categorical data .Results Of the 67 CHB patients with average age of (35 .1 ± 11 .4) ,48 were male and 19 were female;32 were infected with genotype C and 35 were infected with genotype B ;40 were HBeAg‐positive CHB and 27 were HBeAg‐negative CHB ;17 were with high ALT levels and 50 were with low ALT levels .IL‐7 ,Tfh ,IL‐21 and HBV specific‐CTL levels in the peripheral blood of genotype C‐infected patients were (20 .79 ± 4 .82 ) ng/L , (3 .93 ± 0 .82)% ,(24 .77 ± 7 .52) ng/L and (0 .20 ± 0 .04)% ,respectively ,while in genotype B‐infected patients , those were (29 .13 ± 8 .17) ng/L ,(5 .92 ± 1 .92)% ,(39 .94 ± 24 .00) ng/L and (0 .40 ± 0 .06)% , respectively .Levels of IL‐7 , Tfh ,IL‐21 and HBV specific‐CTL in genotype C‐infected patients were significantly lower than those in genotype B‐infected patients (t= 5 .027 ,5 .595 ,3 .553 and 15 .133 , respectively ;all P<0 .01) .Nonspecific CTL ,HBV DNA ,ALT and TBil levels in the peripheral blood of genotype C‐infected patients were all significantly higher than those in genotype B infected‐patients (t=4 .899 ,6 .815 ,2 .763 and 4 .899 ,respectively ;all P< 0 .01) .IL‐7 ,Tfh ,IL‐21 ,HBV specific‐CTL levels in the peripheral blood of HBeAg‐positive patients were significantly lower than those in HBeAg‐negative patients (all P<0 .01) .Nonspecific CTL ,HBV DNA ,ALT and TBil levels in the peripheral blood of HBeAg‐positive patients were all significantly higher than those in HBeAg‐negative patients (all P<0 .05) .IL‐7 ,Tfh ,IL‐21 ,HBV specific‐CTL levels in the peripheral blood of patients with high ALT levels were all significantly lower than those in patients with low ALT levels (all P<0 .01) .Nonspecific CTL and HBV DNA levels in the peripheral blood of patients with high ALT levels were both significantly higher than those in patients with low ALT levels (both P<0 .01) .HBV DNA ,IL‐21 and nonspecific CTL were all correlated with IL‐7 (all P<0 .01) .Conclusion The differences of HBV specific‐CTL and liver function in CHB patients infected with genotype B and C may be correlated with interleukin‐7 induced Tfhcells.

This study examined the effect of ischemia-reperfusion injury on the expression of Pim-3 gene in myocardial tissues and their underlying mechanism. Rat models of myocardial ischemia-reperfusion injury were established by ligating the left anterior descending coronary artery of the rats. A total of 30 SD male adult rats were randomly divided into 5 groups: group A (sham operation, n=6); group B (in which the rats were subjected to 15 min of ischemia by ligation of the left anterior descending coronary artery, n=6); group C (in which the rats received 30 min of ischemia, n=6), group D and group E (in which the left anterior descending coronary artery of the rats were ligated for 30 min and then reperfused for 30 min or 120 min, n=6 in each). The left ventricular tissues were removed immediately after the ischemia-reperfusion injury. Neonatal cardiomyocytes were cultured and treated with different concentrations of H(2)O(2) (0, 5, 10, 20 μmol/L) or tumor necrosis factor-α (TNF-α, 0, 1, 5, 10 ng/mL). The mRNA and protein expression of Pim-3 gene was determined by using RT-PCR, western blotting and immunohistochemistry. Additionally, neonatal cardiomyocytes were transfected with Pim-3 siRNA, and induced to develop apoptosis by using H(2)O(2). The results showed that normal myocardial tissues expressed a quantity of Pim-3 gene mRNA and protein. Ischemia-reperfusion injury could up-regulate the mRNA and protein expression of Pim-3 gene in myocardial tissues. Furthermore, H(2)O(2) but not TNF-α up-regulated the Pim-3 gene expression in cultured cardiomyocytes. And Pim-3 silencing failed to strengthen the H(2)O(2)-inducing apoptosis in cardiomyocytes. It was concluded that ischemia-reperfusion injury up-regulated the Pim-3 gene expression through oxidative stress signaling pathway in myocardial tissues.

Pim kinases contribute to tumor formation and development of lymphoma, which shows enhanced DNA replication, DNA recombination and repair. Endothelial cells^(ECs) express all the three members of Pim kinase gene family. We hypothesized that DNA repair gene would regulate Pim expression in ECs. Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium. The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining. The siRNA fragments were synthesized and transfected by using Lipofectamine LTX. The total cellular RNA was extracted from the cells by using Trizol reagent. cDNAs were quantified by semi-quantity PCR. The effects of LY294002 and wortmannin on RNA stability in ECs were also examined. Our data showed that LY294002 and wortmannin, phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors, increased Pim mRNA expression in ECs without altering the mRNA stability. RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1, respectively. Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs. But etoposide, a nucleoside analogue, which could activate DNA-PKcs and ATM, increased Pim expression in ECs. Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.

Objective To built up the ERP model,measure mode and P300 potential reference standard in mice with vascular dementia(VD),and characterize the P300 potential in mice with VD.Methods Fortyeight mice were randomly divided into a normal group.sham operation group and a VD group.The mice in the Vd group were subject to repetitive ischemia and reperfusion by using the ligation of bilateral common carotid arteries so as to establish the VD model.The behavioral abnormalities were investigated by step-down test and water maze test.The N2 and P3 components of P300 potentials were also recorded.Results It was shown that the learning and memory abilities as reflected by the step down test and water maze test scores were decrease in mice in the VD group when compared with those in the normal group and sham operation group(P<0.05).The N2 and P3 latencies significantly prolonged(P<0.01)and P3 amplitudes decreased(P<0.05)in VD group as well.Conclusions In VD mice,there is a significant prolongation of the P300 potential latency and a significant decrease of learning and memory abilities.Recordings of P300 from unanesthetized mice could be an objective,non-invasive,quantitative and valuable electrophysiological method for studying the cognitive function of VD mice.

Objective: This study aims to analyze the therapeutic effect and prognostic factors of carcinoma of parotid gland (CPG). Methods: Data on 103 CPG patients were retrospectively analyzed. The patients were divided into the simple surgery group (Group One) and post-operative radio-chemotherapy group (Group Two). Kaplan-Meier survival analysis, Log-rank test, and Cox re-gression analysis were employed to analyze the five-year overall survival. Chi-square test was applied to compare the local control rate and recurrence-free survival. Logistic regression analysis was used to determine the correlation between all factors and the local control rate. Results:For all patients, the five-year local control rate, five-year recurrence-free survival rate, and five-year overall survival rate were 71.49%, 69.61%, and 76.10%respectively. The five-year local control ratio (81.96%vs. 61.90%), five-year recurrence-free surviv-al (78.69%vs. 59.52%), and five-year overall survival (88.12%vs. 68.50%) were significantly improved in Group Two compared with Group One. The logistic regression analysis showed that the therapeutic method, T staging, as well as pN(+) neck and tumor differentia-tion were significantly correlated to the five-year local control rate and five-year recurrence-free survival (P<0.01). Cox regression anal-ysis showed that therapeutic method, T stage, as well as pN(+) neck and tumor differentiation were significantly correlated to the five-year overall survival (P<0.01). Conclusion:Post-operative radio-chemotherapy can improve the local control and overall survival rates. This therapeutic method is applicable to patients with T3-4 tumors, as well as pN(+) neck and middle-low differentiation.