Objective To study the rapid detection of mycobacterium tuberculosis resistance to streptomycin by reverse dot blot hybridization technique. Methods The oligonucleotide probes of streptomycin-resistant genes (rpsL and rrs) were prepared and dropped on nitrocellulose membrane. The target DNA fragments of M. tuberculosis clinical isolates were labeled with biotin by PCR amplification, and then hybridized with the oligonucleotide probes on the membranes. PCR-SSCP and PCR-direct sequencing (PCR-DS) techniques were used to detect the target fragment of M.tuberculosis as control. Results In 53 M. tuberculosis clinical isolates, the consistent rate of three detection methods was 100%. Both the SSCP mapping of rpsL and rrs genes and the results of membrane hybridization in 9 drug-sensitive strains were identical to those in M. tuberculosis standard strain H37Rv. Of 44 streptomycin-resistant strains, 33 strains had AAG→AGG mutation at the codon 43 of rpsL gene, 6 strains had A→C mutation at the 513 site of rrs gene, 1 strain had A→T mutation at the 513 site of rrs gene, and the detection rate of the target genes mutation was 90 9%. In 53 M.tuberculosis clinical isolates, 40 resistant strains and 9 sensitive strains to streptomycin could be detected using dot blot hybridization and the consistent rate with the in vitro susceptibility test was 92 6%(49/53). Conclusion The reverse dot blot hybridization technique showed high sensitivity and specificity to detect Mycobacterium tuberculosis resistance to streptomycin. It possessed the simple and rapid characteristics, and could be used to detecte streptomycin-resistant M.tuberculosis clinical strains.

Objectives To understand the mutations of embB genes in M. tuberculosis isolates, and to evaluate their clinical value. Method 102 clinical isolates were identified for their mycobacterial species, and then analyzed their embB genes with PCR SSCP, PCR RFLP, and PCR direct sequencing. Results Mycobacterium tuberculosis strain H 37 R v was used as a control. 102 clinical isolates all had the same 16S rDNA SSCP profiles as M. tuberculosis . Forty one drug sensitive isolates had normal embB SSCP and RFLP profiles. Of 61 ethambutol resistant isolates, 23 (37.7%) displayed abnormal embB SSCP profiles. Eight isolates had abnormal RFLP profiles. All embB mutations situated at codon 306, whose EMB MICs were more than 20 ?g/ml. Eight isolates had ATG to ATA or ATT mutations at codon 306. Thirty isolates had ATG to GTG or CTG mutations at codon 306, whose EMB MICs were more than 30 ?g/ml. Conclusions Ethabutol resistances in some M. tuberculosis isolates were due to mutations on embB genes. PCR SSCP and PCR RFLP method might become a simple and rapid diagnostic test for genotypes of M. tuberculosis ethabutol resistance.

Objective To investigate the relationship between carotid artery intima-media thickness and renal function in patients with diabetes mellitus.Methods 424 patients of type 2 diabetes without dialysis were enrolled in a cross-sectional study.According to their artery intima-media thickness (IMT),the patients were divided into normal group and higher IMT group.All patients according to UAER or 24h urinary protein were divided into normal proteinuria group,micro-proteinuria group and clinical proteinuria group.The biochemical examination,eGFR,and atherosclerotic plaque of different groups were compared.Pearson or spearman correlation was used to analyze the relationship between eGFR,IMT and other parameters.Risk factors for eGFR decline were analyzed by binary logistic regression.Results Compared with normal group,patients in the higher IMT group were older [(63.3±10.2) year vs (52.5 ± 10.6) year,P ＜ 0.05],and underwent longer duration of diabetes [(8.9±6.7) year vs (6.2±5.7) year,P ＜ 0.05].Their level of eGFR was decreased [(75.92±28.00) ml/min vs (91.64±24.05) ml/ min,P ＜ 0.05],while plaque incidence (71.3％ vs 18.3％,x2=112.42,P ＜ 0.01) and prevalence of hypertension (56.4％ vs 29.6％,x2=27.22,P ＜ 0.01) increased.Correlation analysis showed that IMT was positively correlated with age (r=0.503,P ＜ 0.01),duration of diabetes (r=0.204,P ＜ 0.01),24 h urine protein (rs=0.175,P ＜ 0.05),plaque (rs=0.562,P ＜ 0.01),and hypertension (rs=0.193,P ＜ 0.01),but negatively correlated with eGFR (r=-0.307,P ＜ 0.01).Logistic regression analysis showed that age,serum uric acid,24 h urine protein and carotid artery intima-media thickness were independent risk factors for eGFR decline [OR=1.115,95％CI(1.053,1.165),P ＜ 0.001;OR=1.008,95％CI (1.002,1.014),P=0.006;OR=1.492,95％ CI(1.170,1.903),P=0.001;OR=1.619,95％ CI(1.121,2.339),P=0.010].Conclusion Carotid artery intima-media thickness is an independent risk factor for kidney function decline in patients of diabetes.

Objective To investigate the changes of inflammatory factors in the blood serum and their relationship with the immune status of patients with active pulmonary tuberculosis(TB).Methods A total of 97 cases of pulmonary tuberculosis were included from Feb 2003 to Oct 2005,57 of active TB,40 in resting period.Another 41 healthy people were used as normal control.ELISA and APAAP method were used to detect the level of TNF-?, IL-1,IL-6 and the changes of CD_(4),CD_(8)and CD_(4)/CD_(8).Results The levels of IL-1,IL-6,TNF-? were(15.3?1.3),(80.5?7.3) and(77.2?9.8) ng/ml in the normal controls,(33.7?3.6),(293.6?30.5) and(190.7?25.2) in the patients of active TB,and(18.2?2.1),(130.7?14.5),(87.5?10.2) ng/ml in the patients at resting period,which were highest in the patients of active TB.The ratio of CD_(4)and CD_(4)/CD_(8) was(32.3?2.9)% and(0.83?0.17) in the patients of active TB,lower than(48.2?4.4)% and(0.83?0.17) of normal controls.Conclusion The increase of inflammatory factors and decrease of immune activity were the clinical characteristic of patients with active pulmonary tuberculosis,which are of inverse relationship.

Objective To construct recombinant plasmid containing CFP-10 gene of Mycobacterium tuberculosis(MTB).Methods The gene fragment of CFP-10 was amplified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA and cloned to pET-32a(+) vector.The recombinant plasmid pET-32a-CFP-10 was transformed into E.coli BL21 (DE3) and induced by IPTG.Results CFP-10 gene fragment was amplified from genomic DNA of Mycobacterium tuberculosis H37Rv strain,and thepET-32a(+) prokaryotic recombinant plasmid was constructed successfully.The recombinant protein was expressed with the induction of IPTG.Conclusions The prokaryotic expression vector for CFP-10 was successfully constructed and the recombinant protein was highly expressed in E.coli BL21 (DE3),which lays a foundation for its subsequent immunological function study.

OBJECTIVE:To establish the quality standards for Qiqilian capsule. METHODS:TLC was used to identify the As-tragali Radix,Phellodendri chinensis,Coptidis rhizom. HPLC was used to determine the contents of ginsenosides Rg1,ginsenosides Rb1 and notoginsenosides R1. The column was Shim-pack VP-ODS C18 with mobile phase of acetonitrile-water(gradient elution) at flow rate of 1.0 ml/min,detection wavelength was 203 nm,column temperature was 20 ℃. RESULTS:TLC of Astragali Radix,P. chinensis,C. rhizom showed cleer sports and good separation. The linear range was 0.9-9.0 μg for ginsenosides Rg1,0.94-9.4 μg for ginsenosides Rb1 and 0.3-3.0 μg for notoginsenosides R1(r≥0.999 5);RSDs of precision,reproducibility and stability test were lower than 3.0%;recoveries were 96.08%-99.75%(RSD=1.52,n=6),97.03%-99.75%(RSD=1.10,n=6)and 96.38%-98.55%(RSD=0.90,n=6),respectively. CONCLUSIONS:The method is simple,good reproducibility,and can be used for the quality control of Qiqilian capsule.

Objective To investigate the expression and clinical significance of serum microRNA (miRNA) expression profiling in the occurrence and progression of diabetic nephropathy.Methods The miRNA expression profiling was detected by miRNA TaqMan Low Density Array chip from 10 patient with diabetic nephropathy,10 diabetes patients with normoalbuminuria and 10 health control.Real-time quantitative PCR was applied to verify the result of miRNA array in serum samples of 66 patients with diabetic nephropathy (36 patients with microalbuminuria,30 patients with macroalbuminuria),40 diabetes patients with normoalbuminuria and 40 health control.And the relationship of differetial expression with clinical features was analyzed.Results miR-150-5p,miR-155-5p,miR-30e-5p and miR-3196 being validated by real-time quantitative PCR differentially expressed in 3 groups of serum samples from the diabetes patients with microalbuminuria (n=36),with normoalbuminuria (n=40) and health control (n=40) (P ＜ 0.05).Serum miR-150-5p (P=0.005) and miR-155-5p (P=0.006) changed significantly between diabetes patients with microalbuminuria (n=36) and with macroalbuminuria (n=30).Compared with the diabetes patients with microalbuminuria,serum miR-150-5p and miR-155-5p increased by 2.3 and 1.5 times in macroalbuminuria group,respectively.Estimated glomerular filtration rate and urinary albumin excretion rate significantly correlated with serum miR-150-5p and miR-155-5p level.Conclusions miR-150-5p and miR-155-5p may be involved in the process of pathological mechanisms of diabetic nephropathy.Serum miR-150-5p and miR-155-5p may be regarded as potential biomarkers to diagnosis the occurrence and development of diabetic nephropathy.

ObjectiveToestablishadiseasesyndromecombinedanimalmodel,theminiaturepigmodelof chronic myocardial ischemia of phlegm-blood stasis syndrome type , by high fat/cholesterol diet feeding and intravenous injection with VD3 and isoproterenol.Methods Miniature pigs were randomly divided into the control (Ctr) group, high fat/cholesterol diet ( HFC) group and chronic myocardial ischemia model of phlegm-blood stasis syndrome ( CMI) group, 5 pigs in each group .The Ctr group was fed with normal regular chow diet , HFC group was fed with high fat/cholesterol diet , while the CMI group was fed with high fat/cholesterol diet and intravenous injection with VD 3 and isoproterenol .The experiment lasted for 24 weeks.The model establishment and its pathological process of phlegm-blood stasis syndrome were evaluated by examinations of body weight , electrocardiogram, activity, blood lipid, myocardial enzymes, hemorheology, inflammation, cardiac index(CI) and myocardial ischemia size (MIS).Results Compared with the Ctr group, the body weight, heart rate(HR), Total cholesterol(TC), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol ( HDL-C ) , atherosclerosis index ( AI ) , low/middle/high shear rate of whole blood viscosity and reduced viscosity, erythrocyte electrophoresis time(EPT), high-sensitivity C-reactive protein (hs-CRP) and IL-6 levels in the HFC group were significantly increased (P <0.05, P <0.01), while the body weight, heart rate, total ST, ST_average, activity, TC, TG, LDL-C, HDL-C, AI, CK, LDH, cTn-1, low/middle/high shear rate of whole blood viscosity and reduced viscosity, EPT, Casson viscosity(CV), hs-CRP, IL-6, CI and MIS in the CMI group were significantly increased (P<0.05, P<0.01), and APN level in the CMI group was significantly decreased (P<0.05).Moreover, AI, CK, LDH, cTn-1, low/middle/high shear rate of whole blood viscosity , EPT, CI and MIS in the CMI group were significantly higher than those of HFC group (P<0.05, P<0.01), while APN in the CMI group was significantly lower than that of HFC group (P<0.05).Correlation analysis showed that MIS was closely correlated to TC , LDL-C, AI, CK, LDH, cTn-1, APN, high/middle/low shear rate of whole blood viscosity , EPT, CV, hs-CRP and IL-6 (P<0.05, P<0.01).The linear regression analysis also showed that phlegm-blood stasis was closely correlated to TC , LDL-C, AI, CK, LDH, cTn-1, APN, CV, EPT, hs-CRP, and IL-6 ( P <0.01), and further linear stepwise regression analysis showed that the evolution of phlegm-blood stasis was closely related to TC , CK and IL-6.Conclusions Minipig model of chronic myocardial ischemia of phlegm-blood stasis syndrome type can be established by high fat /cholesterol diet feeding and intravenous injection with VD 3 and isoproterenol .Their blood lipid metabolism , hemorheology , myocardial enzymes and inflammatory indexes can be used as external biochemical basis of phlegm-blood stasis syndrome type , which may reflect related biological basis of the traditional Chinese medicine theory of “phlegm and stasis cementation , blood-stasis & toxin causing catastrophe”.

Objective To observe the sensitivity of Wuzhishan, Tibetan and Bama minipigs to exogenous fats.Methods A total of 15 male minipigs including 5 WZS minipigs, 5 Tibetan minipigs and 5 Bama minipigs, were used in this study.The minipigs were intravenously injected with fat emulsion and fed with high-fat diet, and the changes of serum total cholesterol (TC), low density lipoprotein (LDL-C), high density lipoprotein (HDL-C) and triglyceride (TG) levels were detected at 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h and 3 h after intravenous injection of fat emulsion and at 3 h and 12 h, 24 h, 36 h, 48 h, 60 h, 72 h and 84 h after fed with high-fat diet, respectively, and calculated the changes of area under the curve (ΔAUC) values.Results The triglyceride (TG) in the three kinds minipigs were significantly increased (P<0.01), and reached a peak at 0.5 h after injection.The degree of elevation of ΔAUC values were different showing on increasing order: Tibetan minipig > Wuzhishan minipig > Bama minipig, while TC, LDL-C and HDL-C showed no significant changes (P>0.05).Moreover, TC, LDL-C, HDL-C and TG were significantly increased in the three stocks of minipigs induced by feeding with high-fat diet (P<0.05, P<0.01).Among them, the TC and LDL-C of Wuzhishan minipigs peaked at 36 h, HDL-C peaked at 48 h and TG peaked at 24 h after feeding, respectively.TC, LDL-C and HDL-C in the Bama minipigs and Tibetan minipigs peaked at 48-60 h and TG peaked at 36 h after feeding, and the ΔAUC values of TC, LDL-C and HDL-C were in an increasing order of Wuzhishan minipigs > Bama minipigs > Tibetan minipigs.Conclusions The three stocks of minipigs are sensitive to TG after intravenous injection of fat emulsion, and the lipid tolerance values are in an order of Tibetan minipig > Wuzhishan minipigs > Bama minipigs.Meanwhile, the three stocks of minipigs are also sensitive to TC, LDL-C and HDL-C after feeding with high-fat diet, and the lipid tolerance values are in an increasing order of Wuzhishan minipig > Bama minipigs > Tibetan minipigs.

Objective To analyze the genetic variation among white hair black eyes (WHBE) rabbit, Japanese white ( JW) rabbit and New Zealand white ( NZW) rabbit using random amplified polymorphic DNA ( RAPD) technique . Methods Thirty rabbits (male/female 1∶1) of each strain were used in this study.The genomic DNA was extracted from 90 rabbits.Sixty arbitrary primers were used to amplify DNA of rabbits with RAPD-PCR method.Based on the preliminary experiments , polymorphic primers were selected to analyze the genetic variation among the three rabbit strains .The experi-mental data were analyzed using Popgene 3.2 software.Results (1) Twenty-five polymorphic primers were selected among 60 arbitrary primers.493 amplified fragments were detected ranging from 100 bp to 1800 bp.Sixteen primers among 25 arbitrary primers could not only amplify the common DNA bands of 3 rabbit breeds , but also amplify particular alleles in the WHBE rabbit.(2) 234 RAPD sites were detected by agarose gel electrophoresis in WHBE rabbit , among which 166 sites were polymorphic , accounting for 70.94%.228 RAPD sites were detected by agarose gel electrophoresis in the JW rabbit, while 122 sites of them were polymorphic , accounting for 53.51%.231 RAPD sites were detected by agarose gel e-lectrophoresis in the NZW rabbits , with 94 sites being polymorphic, accounting for 40.69%.(3) The Shannon genetic di-versity index of WHBE rabbit, JW rabbit and NZW rabbit was 0.3385, 0.2222 and 0.1905, respectively.(4) The genet-ic similarity between JW rabbit and NZW rabbit was highest among the three rabbit breeds (0.8443), followed by that be-tween WHBE rabbit and JW rabbit (0.8204), and the genetic similarity between WHBE rabbit and NZW rabbit (0.7862) was the lowest .Conclusions Our results demonstrate that there are both genetic similarities and genetic variations among WHBE rabbit, JW rabbit and NZW rabbit .The RAPD technique can be used to delect the genetic relationships among dif-ferent breeds and different individuals of the same breed of rabbits .