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.

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.

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

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 investigate the prevalence of low T3 syndrome in patients with acute myocardial infarction(AMI) and explore the effect of low T3 syndrome on outcome of AMI.MethodsThree hundred and thirty-eight patients with AMI admitted to cardiac care unit(CCU) underwent examinations of thyroid function and cardial ultrasound,and were further categorized according to thyroid hormone profile.The records of noninvasive bi-level positive airway pressure(BiPAP)ventilation utilization,length of hospital stay,mortality during hospitalization were evaluated,and the related factors were analysed.ResultsOne hundred and thirty-nine of the 338 patients(41.12%) with AMI complicated with low T3 syndrome.Free triiodothyronine(FT3) was the independent influential factor for length of hospital stay.Low FT3 was significantly correlated with noninvasive BiPAP ventilation utilization and mortality during hospitalization.The average time of follow-up was(21.4?8.1) months.It was revealed by multivariate Cox regression analysis that FT3 was the chief predictor for cumulative death(risk ratio,4.25;95% confidential interval,2.30-7.87),followed by age and left ventricular ejection fraction.ConclusionThe recognition of AMI complicated with low T3 syndrome plays an important role in predicting the disease severity and outcome.

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.

Background Glaucoma is the second leading cause of blindness,which is characterized by processing retinal ganglion cells (RGCs) loss and optic nerve dystrophy.Clinical study showed that lowing IOP can not arrest the glaucomatous damage of RGCs.To seek a neuroprotective drug is an urgent need.Objective This present study focused on the effect of triptolide,a natural biologically active compound extracted from Tripterygium wilfordii,on RGCs in glaucomatous eyes. Methods Glaucoma animal models were established in the right eyes of 80 clean Wistar rats by combination with aspiration of aqueous humor and phtocoagulation on anterior chamber angle.Wistar rats were assigned to two groups at random.Triptolide (5μg/kg) was intraperitoneally injected daily from three days before photocoagulation through scarification of animals (total 8 weeks),and same amount of physiologic saline solution was used at the same way.IOP was measured with a Tonopen XL tonometer at at 1day,3,5,7 days and weekly for 8-week duration after phtocoagulation.RGCs numbers was calculated by retinal Nissl staining.Morphology of retina in frozen section was examined under the light microscope.The experiment followed the Standard of Association for Research in Vision and Ophthalmology. Results The IOP was elevated in model eyes from 1 day through 3 weeks after operation with statistically different in comparison with before operation(P<0.05).No obvious differences in IOP changg was found hetween the triptolide group and the normal saline group at each time point(P>0.05).The numbers of RGCs of model eyes in normal saline group decreased gradually after operation,but no evident decline of numbers of RGCs in model eyes in triptolide group. RGCs in triptolide group were considerably more than those of normal saline group in various time points after operation ( P<0. 05). However,no obvious difference in RGCs numbers was found between model eyes and control eyes in Triptolide group. Conclusion Triptolide could protect RGCs in glaucomatous eyes,and its effect does not depend on IOP in chronic glaucoma model.

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.