To explore the destructive effect of residual uracil DNA glycosylase on dUTP incorporation to the cDNA PCR products,PAGE and DNA-EIA hybridization technique were applied to detect the efficiency of anti-contamination and the rate of DNA hybridization.In this study,it was found that:⑴Good effect could be achieved with the reaction of 2u of uracil DNA glycosylase(UDG)for 20min at 37℃.⑵UDG could be inactivated at 94℃ for 10min.The hybridization rate of control group of PCR products was 94 18% at room temperature for 65 hours.The hybridization rates of test groups at-20℃ for 65 hours were 87 69%,77 24%,76 83%,respectively.And the inactivation times of anti-contamination groups at 94℃ were“0”min,“10”min and “20”min,respectively;at room temperature for 65 hours,these hybridization rates were 74 77%,72 50%,70 29%,respectively.There were significant differences between control and rest groups(P0 05).Our study suggested that the hybridization rate of test groups kept at-20℃ decreased 19 41%,after 65 hours,the rate decreased 27 45%,but in control group only decreased 5 82% for 65h at room temperature.So that,we conclude that the residual UDP can cause above results.

Remote organ ischemic preconditioning is to conduct a transient and sublethal ischemic adaptation in non-vital organs before occurring cerebral ischemia/reperfusion injury in remote vital organs. Remote organ iscbemic preconditioning has been studied for as long as 15 years in the field of myocardial iscbemia. However, only recently it has become a therapeutic strategy for the treatment of cerebrovascular diseases, This article briefly reviews the methods and mechanisms involved in the protective effects of cerebral ischemia of remote organ ischemic preconditioning.

Objective To investigate the safety,efficacy and effects of emergent percutaneous coronary intervention (PCI) in patients from Second Affiliated Hospital,Medical College,Zhejiang University with cardiogenic shock (CS) complicating acute myocardial infarction (AMI).Method Twenty-seven patients with CS complicating with AMI were treated by PCI with intraaortic balloon counterpulsation (IABP) support.The change of hemodynamics before and after IABP and PCI,the characteristics of PCI,the mortality during hospitalization, the major adverse cardiac events (MACE) and left ventricular ejection fraction at 30-day follow-up were observed.Results The hemodynamics were significantly improved after IABP.No patients died during PCI.Two patients died after PCI and the total mortality was 7.4% in hospital.During the period of 30-day follow-up, one patient died of heart failure.The left ventricular ejection fraction greatly improved at 30 days after PCI. Conclusions The data suggested that the use of IABP during PCI in patients with CS complicating AMI was safe, decreased mortality and improved prognosis.

Objective To explore the mechanism of hepatic autophagy inhibition induced by ischemia-reperfusion injury in the aging liver.Methods The healthy male Lewis rats aged 3 months (3M) and 24 months (24M) were selected,and then were randomly divided into 3M IRI group,3M sham operation group,24M IRI group,24M sham operation group.In the experimental group,noninvasive vascular clamp was used to clamp the left and middle hepatic lobes (about 70o％ hepatic ischemia).The liver was subjected to ischemia at 37 0.5℃ for 30min and reperfusion for 6h.The hepatic duodenal ligament was dissected only by sham operation.The serum levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured at 6 h after operation.Liver tissues of each group were examined by liver pathology and the number of autophagosome of LC3B in liver tissue of each group was observed under confocal microscopy.The changes of autophagyrelated protein were analyzed by Western blotting.Results The levels of serum ALT and AST in 24M IRI group were significantly higher than those in 3M IRI group,the difference was statistically significant (P＜0.05).Pathological analysis showed that 3M IRI group showed spotty necrosis,the 24M IRI group showed massive necrosis and the infiltration of the inflammatory cells;Confocal microscopy showed that the number of autophagosome in the liver tissue of the 24M sham group was slightly lower than that of the 3M sham operation group and the number of autophagosome in the 24M IRI group was significantly lower than that in the 3M IRI group (P＜0.05).The levels of autophagyrelated proteins (Beclin1 and ATG4B protein) in 24M IRI group were significantly down-regulated compare to 3M IRI group (P＜0.05).Conclusion The ischemia-reperfusion injury of liver in aged rats inhibits autophagy,and its mechanism may be related to the decrease of autophagy-related protein level in hepatic ischemia-reperfusion injury.

0.05).The percentage of CD40 positive cells in CBMC-derived DC was lower than that in PBMC-derived DC[(34.80?7.77)% vs(54.37?9.57)%,P

AIM: To study effect of endogenous carbon monoxide on intracellular calcium concentration and explore the mechanism in brain protection of endogenous CO in focal cerebral ischemia in rats. METHODS: SD rats were divided into three groups randomly, which including hemin, ZnPP group and saline group as control. Respectively saline, hemin, ZnPP were injected intra-peritoneally twelve hours before middle cerebral artery was occluded. Twenty four hours after MCAO model was set up, the concentration of carbon monoxide in blood and intracellular calcium in neural cells was examined. RESULTS: Contrast to saline group, the concentration of CO in blood rose up while intracellular calcium in occluded side decreased in hemin group; the concentration of CO in blood went down while intracellular calcium in occluded side rose up in ZnPP group, there was significant difference among them (P0.05). CONCLUSIONS: It may be one of mechanisms on brain protection in ischemic cerebral tissue that carbon monoxide affected intracellular calcium concentration of neural cells by regulating Ca~(2+)-K~+ channel on cell membrane as a messenger gaseous molecular and neurotransmitter. [

Objective　To explore the role of acute infection of Chlamydia pneumoniae (Cpn) in respiratory diseases. Methods　Microimmunofluorescence test was used to detect IgG antibodies for Cpn in serum obtained from 93 inpatients and PCR was used to test Cpn in detection of Cpn DNA in throat specimens from 55 of the 99 patients. Results　Acute Cpn infection was diagnosed in 35.5% of the respiratory diseases. Antibodies for Cpn (titer of ≥512) were present in 47.6% of the pneumonia group, which may suggest that during 1998 to 1999, Cpn caused an epidemic in Beijing. They were also present in 50% of asthma group, 50.0% of pulmonary heart disease group and 26.3% of lung cancer group. Only five patients (9.1%) were positive by PCR. There exists discrepancy between serological and PCR results. Conclusion　Detection of IgG antibodies for Cpn conduces to diagnosis of acute Cpn infection and give advice for appropriate therapy.

Objective To evaluate clinical significance of two real-time fluorescence quantitative PCR kits for quantitative detection of HBV DNA and detection performance at different viral load levels.Methods A series of calibrators with different concentrations(1×106,5×105,1×105,5×104,1×104,5×103,1×103,5×102,1×102,1×101 kIU/L) were prepared with AB-type sera using the second generation WHO international standard (NIBSC code:97/750). HBV viral load in the sera of 78 patients,30 healthy blood donors and 10 calibrators were detected by real-time fluorescence quantitative PCR HBV DNA test kit from PIJI Bio-Technical Development Company Ltd (PG kit) and Cobas AmpliPrep/Cobas TaqMan HBV test kit. The correlation of the two methods was evaluated, and the performance of the two kits different viral load levels was evaluated. The false negative rate was analyzed. Negative control, low positive control and high positive control were included in every batch. Results Both two kits showed the correct results for the 10 specimens from the WHO international standards. The lowest detection limit of HBV DNA for Roche Cobas AmpliPrep/Cobas TaqMan HBV test and PG kit were 2.00 (kIU/L, lg) and 3.00 (kIU/L,lg) ,respectively. There was linear correlation between the results from Roche Cobas AmpliPrep/Cobas TaqMan HBV test and PG kit ( R2=0.938 7, P < 0.01 ), the upper limit of Roche kit had coincided with theoretical value. The samples with HBV DNA level above the upper limit of detection were diluted and retested to obtain the precise result. The result form Roche Cobas AmpliPrep/Cabas TaqMan HBV test [(8.35±0.20) kIU/L, lg] was higher than that from PG kit [(7.73±0.42 ) kIU/L, lg] (t=3. 776, P <0.05) . The detection of 108 serum samples showed that the level of HBV DNA detected by Roche Cobas AmpliPrep/Cobas TaqMan HBV test [(5.88±1.64) kIU/L, lg] was higher than that by PG kit [(5.25±1.55 kIU/L,lg] (t=12. 297 ,P <0.01 ). The correlation coefficients were high in samples with high HBV viral load[HBV DNA(>5.00 and≤7.00) kIU/L,Ig,R2=0. 779 7, P <0.01 ;HBV DNA( >7.00 ands≤9.00) kIU/L,lg,R2=0.603 7, P <0.01]. The correlation coefficient was low in samples with low HBV viral load[HBV DNA ( > 3.00 and≤5.00) kIU/L, lg, R2=0. 417 3, P <0.01 )]. When HBV DNA ( >3.00 and≤4.00) kIU/L,lg,the false negative rate was 33.3% (5/15). When HBV DNA ( > 1.08and≤3.00) kIU/L,lg,none of positive samples was detected with PG kit. Conclusions PG kit is not as good as Cobas AmpliPrep/Cobas TaqMan HBV test . The linear correlation between the results from the two kits is good. The correlation between the results detected with PG kit and Cobas AmpliPrep/Cobas TaqMan HBV test is higher in the high viral load groups than in the low viral load group. It is suggested that PG kit had a narrower linear range.

Objective To determine the conversion equation for X(copies/ml, lg) quantity values of domestic HCV RNA quantitative fluorescence amplification assays approved by SFDA and Y( IU/ml, lg)reference values of standard substance. Methods The second generation WHO International Standard (NIBSC code:96/798) was mixed with human AB blood type serum to create 7 different dilutions which included 100 000, 50 000, 25 000, 10 000, 5 000, 2 500 and 1 000 IU/ml. Two different batches of each three domestic hepatitis C virus RNA real-time fluorescence quantitative PCR assays and 2 different batches of each assay were employed to detect the 7 different concentration samples with real-time PCR. Each test was performed 4 times repeatedly. Results The correlations between X( copies/ml,lg) values of domestic HCV RNA assays and Y(IU/ml,lg) reference values of standard substance were as follow,Assay A:Y =0. 902 0 X+0.284 9,R2 =0.953 3,P＜0. 01,n =56;Assay B: Y=0. 875 7 X +0.562 4,R2 =0.956 5,P＜0.01,n =56; Assay C: Y = 0. 843 8 X + 0. 560 5, R2 = 0. 945 8, P ＜ 0. 01, n = 56. Conclusions All the conversion equations are different among the quantity value of three assays and the reference values of standard substance, that suggests it is necessary to perform more stringent traceability analysis for the quantity values of 3 assays. Through standardizing the quantity values preliminarily, the conversion equation can enhance the comparability between the quantity values of different assays, and provide a standard of HCV RNA virus load detection for clinical diagnosis and treatment monitoring of HCV infection.