Objective To investigate the isoniazid-dependence of clinical isolated strains of M.tuberculosis.Methods The double-phase Kuang's agar medium was employed to isolate M.tuberculosis from the patient's sputum,and its drug-resistance and drug-dependence were determined.Results 184 clinical isolates of isoniazid-dependent M.tuberculosis were grouped as follows.Type A:the strains grew faster and more vigorous in drug tubes than in control tubes,and there were 12 strains(6.5%)which grew more colonies in high concentration tubes than in low concentration tubes;Type B:the strains grew faster and more vigorous in drug tubes than in control tubes,and there were 10 strains(5.4%)which grew more colonies in low concentration tubes than in high concentration tubes;Type C:the strains grew faster and more vigorous in low concentration tubes than in control tubes,and there were 15 strains(8.15%)which grew less colonies in high concentration tubes than in control tubes;Type D:the strains grew faster and more vigorous in high concentration tubes than in control tubes,and there were 4 strains(2.2%)which grew less colonies in low concentration tubes than in control tubes.The isoniazid-dependent strains covered 22.3%(41/184)of the total clinical isolates.Conclusion The isoniazid-dependent strains did exist in the clinical isolates of M.tuberculosis,and different features of dependence have been detected.

Objective To investigate the feasibility of multiplex PCR for rapid identification of Mycobacterium tuberculosis and non-tuberculosis Mycobacteria. Methods According to MTP40 gene sequence of Mycobacterium tuberculosis, 32kD gene sequence of Mycobacterium and IS6110 insertion sequence gene sequence of Mycobacterium tuberculosis complex, three specific pairs of primers (PT1-PT2, MT1-MT2 and IS5-IS6) for Mycobacterium were designed, and the target DNA for MTP40, 32kD and IS6110 was 396bp, 506bp and 984bp, respectively. The genome of 92 Mycobacterium tuberculosis clinically isolated strains and 5 non-tuberculosis Mycobacteria clinical strains were amplified in the same system, and the results were compared with reference strains. Results Among 92 clinical strains of Mycobacterium tuberculosis, the DNA fragments of 396bp, 506bp and 984bp were found in 90 Mycobacterium tuberculosis clinical strains, as well as in the reference strain H37Rv; the sensitivity of multiplex PCR for Mycobacterium tuberculosis was 97.8%, and the specificity was 100.0%. The DNA fragments of 506bp were all found in 5 non-tuberculosis Mycobacteria clinical strains, the sensitivity and specificity for non-tuberculosis Mycobacterium were both 100.0%. Conclusion The multiplex PCR is a rapid, sensitive and specific method for identification of Mycobacterium tuberculosis and non-tuberculosis Mycobacteria, and it may provide an effective way for clinical diagnosis of Mycobacterium tuberculosis and non-tuberculosis Mycobacteria, therefore useful in clinical application.

ObjectiveTo detect specific antigens of neural cells in amniotic epithelial cells(AECs) in rats. MethodsAECs were dissociated and purified from the amnion of pregnancy 12—14 d rats. The expression of specific markers of neural stem cells (Nestin, Musashi) and differentiated cells (MAP-2,NSE,GFAP) and ChAT, NT-3 in the AECs were detected by immunocytochemistry. ResultsThe cultured AECs displayed positive immunoreactivity to MAP-2, NSE, GFAP, Nestin and Musashi. In addition, the cells also demonstrated immunoreactivity to ChAT and NT-3. ConclusionAECs are similar with neural cells and it may be useful as a sustained source to improve outcome of neural stem cells transplantation.

ObjectiveTo investigate an effective method to isolate neural stem cells(NSCs).MethodsNSCs were dissociated by digestion with trypsin, EDTA and different doses of Dispase,and serum-free culture techniques and immunohistochemistry techniques were used to verifying the dissociated.ResultsA lot of single neural stem cells were obtained by using Dispase to digest neurosphere, and the cells could keep its structure and morphology.ConclusionIt is an ideal method by using Dispase to digest neurosphere for isolating NSCs.

Adult ; China ; Humans ; Male ; Neisseria meningitidis, Serogroup C ; drug effects ; genetics ; isolation & purification

200 papers on nerve root type cervical spondylosis treated with Chinese medicine were retrieved and 38 papers with complete diagnostic criteria and medical statistics were included for study. The results showed acupuncture, massage, and herbal therapy were three common methods and have their own advantage, but systemic, standardized and normative treatment program was lack. In the meantime of treating nerve root type cervical spondylosis, prevention should also be paid attention. The treatment, prevention and exercise on the whole therapeutic idea should be established, which has far-reaching significance.

Objective To analyze the dielectrophoresis patterns of the proteome of the Rifampin-dependent and-resistant stains of Mycobacterium tuberculosis,to search and identify the differently expressed proteins,and to provide the proteomic basis for researching the mechanism of anti-tuberculosis drug dependence of M.tuberculosis.Methods The whole somatic proteins were extracted from two strains of M.tuberculosis.The first dimensional ampholine electrophoresis was performed on immobilized pH gradient(IPG) rod gels(pH 4-7).Then the proteins on IPG strips were separated using SDS-PAGE.The stained gels were scanned with image scanner and the images were analyzed by Imagemaster 2D software.The differentially expressed proteins were detected by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS).Results Seven hundred and fifty-three spots were detected in Rifampin-dependent strain of M.tuberculosis,while 584 spots were detected in Rifampin-resistant strain,including 404 match spots(the match rate: 61.5%).As to the expression in Rifampin-dependent strain,7 spots significantly up-regulated and 35 spots down-regulated,6 spots were absent in expression,and 5 spots expressed separately,most of the spots were small molecular proteins.Ten spots were selected to run MS analysis.Nine spots were identified as representing 7 proteins.Conclusion The Rifampin-dependent strain of M.tuberculosis is characterized by a rapid and vigorous growth mainly by means of the differential expression of enzymes related to energy metabolism and fatty acid biosynthesis.

Three strains having degrading poly(?-hydroxybutyrate)(PHB) activity were isolated from activated sludge of different ecological environments and areas,named DS9701, DS9710 and DS9713.The properties of PHB depolymerase produced by DS9701, DS9710 and DS9713 were studied. All the PHB depolymerases are extracellular enzyme and are induced enzyme. The time that enzyme activities of the PHB depolymerases reach the maximum is 96 hours after inoculation. The apparent optimal temperature range for crude enzymes extract is 40℃～45℃.

A multiplex real-time PCR was developed to detect ctxA of Vibrio cholerae, gyrB and tdh of Vibrio parahaemolyticus simultaneously. The multiplex real-time PCR were evalidated by detection for the three genes in 47 toxigenic V. cholerae O1 and O139 strains (ctxA+; O1=3, O139=44), 25 non-toxigenic V. cholerae strains (ctxA-; O1=12, O139=6, non-O1 and non-O139=7), 116 V. parahaemolyticus strains with or without tdh (73 or 43) and 9 other bacteria strains. The specificity and sensitivity of the multiplex real-time PCR in detection for the ctxA and the tdh genes in the strains tested were both 100.0%, compared to the results by routine PCRs. In the detection for V. parahaemolyticus specific gyrB using the multiplex real-time PCR, all of 116 V. parahaemolyticus strains were positive, and 9 other strains and 72 V. cholerae strains were all negative. The multiplex real-time PCR is a sensitive, specific and quick assay not only for detecting virulence genes of V. cholerae and V. parahaemolyticus but also for identifying V. parahaemolyticus at species level. In addition, two real-time PCRs for detection of V. parahaemolyticus virulence genes trh1 and trh2 were also developed.

Objective To study the cell vitality and ultra-structure of in vitro cultured fetus chondrocytes exposed to different doses of fluoride.Methods Primary chondrocytes were obtained from articular cartilage of the 24-27 weeks,aborted and dead fetuses.The third generation of primary cultured chondmcytes were exposed to concentrations of 0,10-2,5 × 10-3,10-3,10-4,10-5,10-6,10-7 and 10-8 mol/L fluoride for 24,48 and 72 h.Cell vitality was detected with Cell Counting Kit-8 (CCK-8) and ultra-structure of chondrocytes was observed by transmission electron microscope.Results The cell vitalities of chondrocytes exposed to doses of fluoride (10-2,5 ×10-3,10-3,10-4,10-5,10-6,10-7 and 10-8 moL/L) for 24,48 and 72 h were(15.04 ± 0.55)％,(62.53 ± 1.03)％,(100.34 ± 5.19)％,(111.40 ± 3.69)％,(121.47 + 6.09)％,(129.95 ± 4.96)％,(121.81 ± 4.97)％,(111.00 ± 1.63)％;(10.35 ± 0.64)％,(35.23 ± 2.41)％,(110.30 ± 2.07)％,(113.66 ± 6.98)％,(120.36 ± 6.23)％,(133.40 ± 5.80)％,(126.06 ± 5.40)％,(115.62 ± 7.33)％; (6.19 ± 0.16)％,(18.44 ± 0.21)％,(120.83 ± 4.93)％,(123.77 ± 4.82)％,(129.09 ± 5.21)％,(140.44 + 4.18)％,(131.99 ± 7.00)％,(124.10 ± 3.68)％,respectively.The cell vitalities of 10-2,5 × 10-3 mol/L fluoride groups were significantly lower than that of the control group (all P ＜ 0.05).The cell vitality of 10-2 mol/L group was significantly lower than that of the 5 × 10-3 mol/L group (P ＜ 0.05).Doses of fluoride (10-2,5 × 10-3 mol/L) could inhibit the cell vitality and promote the apoptosis of chondrocytes in vitro with increasing doses and prolonged time.The cell vitalities of 10-3,10-4,10-5,10-6,10-7,10-8 mol/L of fluoride groups were significantly higher than that of the control group (except the 24 h 10-3 mol/L,P ＜ 0.05).Between 10-4 and 10-3 mol/L groups(the vitalities of 48 h and 72 h were higher,but not significantly); 10-5 and 10-4 mol/L groups (the vitality of 72 h was higher,but not significantly); 10-6 and 10-5 mol/L groups,the cell vitalities were significantly higher than that of the control group(all P ＜ 0.05).Between 10-7 and 10-6 mol/L groups,10-8 and 10-7 mol/L groups (the vitality of 72 h was lower,but not significantly),the cell vitalities were significantly lower than that of the control group(all P ＜ 0.05).Doses of fluoride(10-3-10-8 mol/L) could promote the cell vitality of chondrocytes in vitro with prolonged time.The optimal concentration for the promotion was 10-6 mol/L.The cells of the control group were characterized as regular morphology,the abnormal surface microvillis,abundant cytoplasm and mitochondrial,abundant and slightly expanded rough endoplasmic reticulums and low electron-dense materials.The cells of 10-6 mol/L fluoride group had the following changes,increased and swell mitochondrial,hypertrophy and expanded rough endoplasmic reticulums.The cells of 5 × 10-3 mol/L fluoride group had the following changes,decreased microvillis,invaginated cell membrane,pyknosis and apoptotic body.Conclusion Doses of fluoride (10-3-10-8 mol/L) can promote the proliferation of human chondrocytes cultured in vitro.Doses of fluoride (10-2,5 × 10-3 mol/L) can promote the apoptosis of human chondrocytes cultured in vitro.