Objective　To investigate the changes of plasma epinephrine (E) and norepinephrine (NE) in rabbits with firearm thoracic wounds. 　Methods　 A total of 24 rabbits were divided into 3 groups randomly, the simple thoracic penetrating injury group (Group A, n = 8), the pseudo-injury group (Group B, n = 8)and the normal control group ( Group C, n = 8). The pressures of carotid artery and jugular vein were recorded by eight-road physiological recorder at 5 minutes before injury. And the concentrations of plasma E and NE were detected through high-performance liquid chromatography (HPLC) and electrochemical detection (ED) at 5 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours and 24 hours after injury in all the rabbits. 　Results　The concentrations of plasma E and NE in Group A were significantly higher than those of Group C (P<0.01) and changed regularly. There was no significant difference between Group B and Group C. The pressures of carotid artery and jugular vein of Group A increased by 1.8 and 6.8 times, respectively, during 2 millisecond after injury, then decreased to normal immediately. They decreased to the minimum (0.63 and 0.25 time, respectively) in 30 minutes and 5 minutes respectively and averagely, and raised to normal slowly after 6 hours and 12 hours respectively and averagely. There was no significant difference between Group B and Group C.　Conclusions 　 The concentrations of plasma E and NE increase significantly in the rabbits with thoracic firearm wounds. Their changes have close relationship with the severity of wound and can be used as a judging criterion during the early period of wound.

Objective To discuss clinical application of theory of TCM constitution on non-alcoholic fatty liver disease (NAFLD).Methods Literature of nearly 15 years was summarized to analyze the development of Chinese constitution theory and its application of on typing and treatment of NAFLD.Results Main types of constitution in patients with NAFLD were phlegm-dampness,deficiency of Qi and damp-heat.On tiis basis,treatment based syndrome differentiation of TCM could effectively prevent and treat NAFLD.Conclusion TCM constitution theory provide research ideas for NAFLD.

To identify the species of Mycobacterium clinical isolates by molecular biology techniques,six clinical isolates which were preliminarily recognized as Mycobacterium tuberculosis by the TCH and PNB culture methods were selected in this study.PCR was applied to amplify the oxyR-ahpC interval resistant-zone(intergenic region) and the length of PCR product in four strains was the same as that of H37Rv,while the length in the other two strains was different from that of H37Rv but same as Mycobacterium intracellulare 95002.With sequencing and on-line homology comparison with H37Rv,U18263 and U71061,the DNA sequence of oxyR-ahpC intergenic region displayed a 99% homology with Mycobacterium intracellulare U71061 and an 84% homology with Mycobacterium tuberculosis H37Rv.In addition,the results of hsp65 PCR-restriction fragment length polymorphism analysis and multi-locus PCR amplification in the two strains were identical with those of Mycobacterium intracellulare 95002.These two clinical isolates which were preliminarily recognized as Mycobacterium tuberculosis by PNB and TCH culture methods were finally identified as Mycobacterium intracellulare.Results predicted that the application associated with various techniques of molecular biology would provide a faster,easier and more correct way for the species identification of Mycobacterium.

Objective: To examine the association between the cross-resistance to ethionamide (Eto) and isoniazid (INH) and mutations of drug resistant genes in Mycobacterium tuberculosis (MTB), so as to provide the evidence for clinical diagnosis and treatment for multidrug-resistant (MDR) tuberculosis. Methods: Totally 126 MTB clinical isolates were selected, including 88 MDR-MTB clinical isolates and 38 INH- and rifampicin (RFP)-sensitive isolates. The resistance to INH and Eto was tested in MTB clinical isolates using the drug susceptibility test, and the mutations in the spacer region of INH and Eto resistance-related katG, inhA, ethA, mshA, ndh, spacer region of oxyR-ahpC and inhA promoter were detected using PCR assay. The phenotypic resistance served as a gold standard, and the sensitivity, specificity and accuracy of gene mutation tests were calculated for detection of MTB clinical isolates cross-resistant to INH and Eto. Results: Of the 126 MTB clinical isolates, there were 37 isolates cross-resistant to INH and Eto (29.37%), 51 isolates with resistance to INH and susceptibility to Eto (40.48%), 4 isolates with susceptibility to INH and resistance to Eto (3.17%) and 34 isolates with susceptibility to INH and Eto (26.98%). Among the 41 Eto-resistant MTB clinical isolates, there were 37 isolates with resistance to INH (90.24%). There were 64 MTB clinical isolates detected with katG mutations (50.79%), 4 isolates with mutation in the spacer region of oxyR-ahpC (3.17%), 2 isolates with inhA mutations (1.59%), and these isolates were all resistant to INH. There were 11 MTB clinical isolates detected with mutation in the inhA promoter (8.73%) and one isolate with ndh mutation, and all these isolates were cross-resistant to INH and Eto. There were 23 MTB clinical isolates detected with ethA mutations (18.25%) and 40 isolates with mshA mutations (31.75%), in which Eto-susceptible and -resistant isolates were detected. The diagnostic sensitivity, specificity and accuracy of inhA promoter tests for detection of cross-resistance to INH and Eto were 29.73% (95%CI: 16.44%-47.17%), 100.00% (95%CI: 87.36%-100.00%) and 63.38% (95%CI: 51.76%-73.63%) in MTB clinical isolates. Conclusions The prevalence of INH resistance is high in Eto-resistant MTB clinical isolates. Mutation in the inhA promoter region correlates with the cross-resistance to INH and Eto in MTB clinical isolates, and detection of mutation in the inhA promoter may be feasible to detect the cross-resistance to INH and Eto in MTB clinical isolates.

Objective To examine the foasibility of a new method for Mycobacterium tuberculosis (M. tuberculosis)"Beijing family"strain identifjcation——RD105 deletion test. Methods Two methods,Spoligotyping and RD105 deletion test,were used for M. tuberculosis"Beijing family"strain identification,respectively. The difference of the two identification methods was compared. Results Three hundred and forty-two clinical isolates from four areas(Beijing,Fujian,Xinjiang and Jilin)were assayed in this study.Among the total samples,261 isolates belonged to"Beijing family"accounting for 76.32%,while the other 81 isolates belonged to"non-Beijing family"accounting for 23.68%. The coincidence rate for these two methods was 100%. Conclusion The simple and rapid new method——RD105 deletion test can be used to identify"Beijing family"instead of the traditional method——Spoligotyping.

Objective To investigate and evaluate the PCR-RFLP method for identification of Mycobacterium abscessus (M.abscessus) group.Methods 46 clinical acid-fast bacilli (AFB) isolates from Pulmonary Hospital of Fujian Province,Center for Disease Control and Prevention of Zhaoyang District in Beijing and Beijing People Liberation Army General Hospital were collected in 2009 -2010 and identified by traditional bacteriological characteristics according to clinical laboratory handbook of mycobacteria (2004).The PCR-RFLP method was used for species identification of M.abscessus group using hsp65 (441 bp) or rpoB (380 bp) gene fragment as specific target,while the direct DNA sequencing was performed as a control method.Results Of 46 AFB isolates,30 strains were identified as Mycobacterium tuberculosis by its traditional bacteriological characteristics and 16 strains were identified as non-tuberculosis mycobacterium (NTM).10 strains of the NTM strains had identical bacteriological characteristics with the reference strain M.abscessus ATCC 19977.Identified by hsp65 PCR-RFLP,9 of these 10 strains got the same pattern of 235bp and 200 bp(BstE Ⅱ )/145 bp,70 bp,60 bp,55 bp,50 bp and 40 bp(Hae Ⅲ ),and 1 got pattern of 235 bp and 200 bp(BstE Ⅱ )/200 bp,70 bp,60 bp and 50 bp(Hae Ⅲ ).While identified by rpoB PCRRFLP,all 10 strains got the same pattern of 105 bp,95 bp and 80 bp( Msp Ⅰ )/130 bp,100 bp and 90 bp (Hae Ⅲ ).By analysis of the DNA sequence,hsp65 and rpoB sequence of these 9 strains showed 100％similarity with those of M.abscessus,while the hsp65 and rpoB sequence of the other one strain showed 100％similarity with those of M.massiliense.Conclusion PCR-RFLP is a rapid and effective method for species identification of M.abscessus group,and hsp65 PCR-RFLP is more effective than rpoB PCR-PFLP in the species identification of M.abscessus group.

The Mycobacterium chelonae/abscessus (M.chelonae/abscessus) complex belongs to the rapidly growing genus Mycobacterium (RGM).It is one of the most important pathogenic members of Mycobacterium leading to nosocomial infections and outbreaks.It includes members of M.chelonae,M.immnunogenum,M.abscessus,M.massiliense,and M.bolletii.In order to investigate the epidemiological characteristics of the M.chelonae/abscessus complex in China and to conduct the molecular methods for species identification of M.chelonae/abscessus,we collected clinical M.chelonae/abscessus complex strains identified by phenotypic tests.Members were verified by sequencing of 16S rRNA,Species and subspecies were identified by hsp65 and rpoB PCR RFLP methods.In total,27 clinical specimens were identified as Mycobacterium chelonae/abscessus complex by phenotypic tests.16s rRNA gene sequence analysis of all 27 clinical samples shared over 99.7％ similarity with M.chelonae and M.abscessus.Species identification with hsp65 PCR-RFLP and rpoB PCR-RFLP revealed that 18 specimens were M.abscessus and 4 were M.absecces.The remaining 5 samples displayed a pattern that failed to match any previously reported pattern.Thus,this might represent a novel species that is part of the Mycobacterium chelonae/abscessus complex.We identified that a majority of the chronic lung infection in China is caused by the M.chelonae/abscessus complex.Specifically,the M.abscessus species might be the most infectious,while other species in the complex can still cause infection.Interestingly,there may be a novel or previously unidentified species that is a part of the complex.Finally,we show that species identification can be carried out more accurately by combined use of hsp65 and rpoB PCR-RFLP.

OBJECTIVE To discover diversity of X gene sequences of hepatitis B virus isolates in hepatitis B induced liver cirrhosis patients and HBV carriers.METHODS DNA fragment including X gene sequences of hepatitis B virus was amplified,sequencing PCR products was preformed.The PCR products of three liver cirrhosis patients and three HBV symptomless carriers were cloned into pGEMT Easy vectors.Positive clones with target sequences were selected out for sequencing.Sequence comparison was made to find the identity.RESULTS A comparison of T1762/A1764,G1719T,T1727G/A,G1730C and T1753C mutations in a core promoter between the liver cirrhosis patients and the HBV carriers showed that the HBV isolates from the former had higher frequencies of mutation than the latter.The X promoter region of the HBV isolates from the liver cirrhosis patients showed higher frequencies of mutation than the isolates from the HBV carriers.Additionally,the homology between clones of X gene from one individual with liver cirrhosis averages 91.3-99.7%,that of HBV carriers averages 96-100%.CONCLUSIONS The core and X promoter region of the HBV isolates from the liver cirrhosis patients show the higher frequencies of mutation than the isolates from the HBV carriers.There are HBV quasispecies which possess great variation in the liver cirrhosis patients.

Objective To understand the status of intestinal parasitic infections and the related knowledge and behavior in residents of Jiaodong area of Shandong Province,so as to provide the evidence for making an appropriate preventive and control strategy. Methods A total of 18 villages from 6 counties in Jiaodong area were selected as investigation sites according to the stratified sampling method. The feces samples of the permanent residents aged above 3 years were collected and examined by Kato-Katz technique to find the intestinal parasite eggs,and the children under 12 years old were examined by the method of cellophane anal swab to detect the Enterobius vermicularis eggs. In addition,50 households in each survey sites were randomly selected to in-vestigate the basic family situation and the condition of awareness on prevention knowledge and formation of correct behavior of res-idents by using a structured questionnaire. Results Totally 6 163 residents involved in the feces examinations,and the total in-fection rate of intestinal parasites was 6.91%. The infection rates of Trichuris trichiura,Ascaris lumbricoides and hookworm were 6.56%,0.62%and 0.21%,respectively. The infection rate of E. vermicularis in children under 12 years old was 0.51%. The eggs of Clonorchis sinensis and Taenia solium were not found in this survey. The awareness rate of knowledge about preventing parasitic diseases was 49.54%. The formation rates of washing hands before eating,washing hands after using the toilet,never eating raw fruit and vegetable without washing clean,never working in the field with bare feet,and never drinking unboiled water were 97.78%,91.95%,88.81%,92.42%and 86.48%respectively. Conclusions The infection rate of intestinal parasites is low in Ji-aodong area,but there is a significant difference among different counties. The awareness rate of knowledge about preventing para-sitic diseases is low,but the formation rate of healthy behavior is high. In the future,the health education and the strategy of tak- ing medicine among the key population should be enhanced,and the project of reconstructing safe water supply and lavatory should be advanced.

Objective To investigate the cross-reactive immune responses to Mycobacterium vac-cae (M. vaccae), Mycobacterium tuberculosis (M. tuberculosis, H37Rv) and Mycobacterium bovis Bacillus Calmette-Guerin ( BCG) for providing reference for the development of new vaccines with M. vaccae. Meth-ods M. vaccae (ATCC95051), M. tuberculosis (H37Rv) and BCG (China strain) were cultured on L-J solid media and harvested. Total bacterial protein antigens prepared by ultrasonic disruption were used to im-munize BALB/c mice. IgG antibodies in serum samples were detected with enzyme-linked immunosorbent assay ( ELISA) to evaluate humoral immune responses. Cellular immunity was assessed by detecting various cytokines with cytokine release assay ( CRA) . Results The mice that were respectively immunized with the three mycobacterial antigens could produce high titers of antibodies ( IgG) and high levels of IFN-γand IL-2, but low levels of IL-4 and IL-10. Results of the cross reactivity tests showed that ATCC95051, H37Rv and BCG were able to cross-react with the immunized mice, and all of them induced high levels of IFN-γ, IL-2 and IgG antibodies. Conclusions The three Mycobacteria mainly elicited Th1 immune responses. There were cross-reactive immune responses to M. vaccae, M. tuberculosis and BCG, which might provide ref-erence for using M. vaccae in the development of new anti-tuberculous vaccines.