Objective To construct mycobacterial membrane-anchored expression vector and to analyze expression level and sub-cellualr localization of exogenous target protein. Methods Based on the mycobacterial intracellular expression vector pMFA42 which contained a strong promoter of pfurAma mutant, the signal sequence of Mycobacterium tuberculosis(Mtb) 19×103 lipoprotein (19SS) was synthesized and was then cloned into the downstream of pfurAma mutant to generate the mycobacterial membrane-anchored expression vector pMFA42M. The coding gene of enhanced green fluorescent protein(EGFP) was amplified by PCR, and then sub-cloned into these two vectors described above to construct recombinant EGFP fused and membrane-anchored strains, respectively. The coding genes of Mtb immuno-dominant antigens Ag85A and its chimera Ag856A2 were then sub-cloned intothe membrane-anchored construct pMFA42MG to produce recombinant Mtb antigen EGFP fused-expression strains. After that, expression levels and sub-cellualr localization of exogenous target protein were further analyzed by Western blot and flow cytometry sorting(FCS), and the fluorescence intensities of recombinant EGFP- expressed strains were observed in vitro directly and after transfection of murine macrophage cell line RAW264.7. Results The novel mycobacterial membrane-anchored expression vector was constructed successfully by introduction of signal sequence of Mtb 19×103 lipoprotein. Using of EGFP as model antigen, exogenous target protein was demonstrated to be expressed with high level and could be anchored into cell membrane of recombinant mycobaterial strains. Conclusion A novel mycobacterial membrane-anchored expression vector was constructed successfully to research recombinant BCG and functions of mycobacterial membrane proteins, and the constructed EGFP-expressed recombinant strains could also be used to research cytophagy in cell model and mycobacterial colony and translocation in animal immunization as model indicator bacteria.

Adolescent ; Adult ; Anemia, Hemolytic, Autoimmune ; drug therapy ; Female ; Humans ; Immunosuppressive Agents ; therapeutic use ; Male ; Middle Aged ; Sirolimus ; therapeutic use ; Treatment Outcome ; Young Adult

BACKGROUND: Apoptosis secondary to ischemia and hypoxia is the main cause of spinal cord dysfunction. Because of the decrease in atmospheric pressure, patients living on the Qinghai-Tibet Plateau are in a hypoxic environment, which is very unfavorable for the recovery of spinal cord injury. Hyperbaric oxygen therapy can improve the postoperative function of patients with incomplete spinal cord injury, and its effect is better on the plateau than at normal altitudes.OBJECTIVE: To observe the effect of hyperbaric oxygen therapy on traumatic spinal cord injury in patients living on the Qinghai–Tibet Plateau.METHODS: This prospective, open-label, randomized controlled clinical trial was performed at the Department of Spine Surgery, Affiliated Hospital of Qinghai University, China. In total, 164 patients with incomplete traumatic spinal cord injury were equally and randomly assigned to a control group and a hyperbaric oxygen therapy group. Patients in the control group were treated with pedicle screw fixation and decompressive laminectomy. In addition to the surgical treatment performed in the control group, patients in the hyperbaric oxygen group underwent hyperbaric oxygen therapy at 0.2 MPa once a day for four treatment courses. Ten treatment sessions constituted one course, and each course was separated by a 5- to 7-day rest interval. The primary outcome was the modified Barthel index to assess activities of daily living. The secondary outcomes were the American Spinal Injury Association (ASIA) impairment scale grade, sensory score, and motor score. The study protocol was approved by the Ethics Committee of the Affiliated Hospital of Qinghai University, China (Approval number: QHC011K). Written informed consent was provided by a relative or legal representative of each patient after they had indicated that they fully understood the treatment plan. RESULTS AND CONCLUSION: The partial results demonstrated that after four treatment courses (55-61 days), the modified Barthel index and ASIA tactile, pain, and motor scores were higher in the hyperbaric oxygen group than in the control group. The ASIA grades were significantly different between the hyperbaric oxygen group and control group. The proportion of patients with ASIA grades D and E was higher in the hyperbaric oxygen group than in the control group. In this trial, we aim to determine the efficacy of hyperbaric oxygen therapy on the treatment of incomplete traumatic spinal cord injury in patients living on the plateau and to provide clinical evidence for treating incomplete traumatic spinal cord injury in these patients.

Objective To investigate the protection of Liuwei Dihuang Jiawei Capsula(LDJ Capsula,Rehmanniae Capsula of Six Ingredients) on diabetic nephropathy(DN) rats′ kidney and effect on renal protein kinase C(PKC) activity and connective tissue growth factor(CTGF) of DN rats.Methods The DN rat models were induced by ip injection of streptozotocin(STZ).The rats were randomly divided into five groups: control group; DN model group;Lotensin group;LDJ Capsula group;and Lotensis and LDJ Capsula combination group.Drug intervention term was 12 weeks.Renal ultrastructure was observed by transmission electron microscope and Masson staining.Relative kidney weight,blood glucose level,serum and urine creatinine content,creatinine clearance,excretion rate of the 24 hour urine protein,renal PKC activity,and CTGF expression in renal cortex were measured by immunohistochemistry.Results Deposition of collagen in renal of DN rats was conspicuous.Relative kidney weight,blood glucose level,serum and urine creatinine content,creatinine clearance,excretion rate of 24 h urine protein,renal PKC activity and CTGF expression of DN rats increased obviously.All Lotensin,LDJ Capsula,and the combination of these two drugs could decrease renal PKC activity and CTGF expression and ameliorate proteinuria and renal function of DN rats.At the same time they all could abate the deposition of collagen in renal of DN rats.Combination of these two drugs could decrease renal PKC activity and CTGF expression more ob-viously and at the same time had more notable protective effect on kidney of DN rats.Conclusion All Lotensin,LDJ Capsula,and the combination of these two drugs could protect kidney of DN rats.The combination of these two drugs has more obviously protective effect than using Lotensin only.

OBJECTIVETo assess the effectiveness and safety of traditional Chinese herbal medicine (TCHM) for chemotherapy-induced leucopenia in patients with malignant tumor.

METHODSChinese database (CNKI, VIP, CBM, Wanfang Database) and English database (Medline, Cochrane Library) were retrieved with the deadline of September 2013. Participants were cancer patients confirmed by pathology waiting for chemotherapy. We included randomized clinical trials (RCTs) testing chemotherapy plus TCHM vs. chemotherapy plus placebo, chemotherapy alone, conventional treatment, or TCHM plus chemotherapy combined with conventional treatment vs chemotherapy combined with conventional treat ment. The primary outcomes were WBC count, leucopenia incidence, and adverse reactions. Assessments of methodological quality, including randomization, allocation, concealment, blindness, dropping-out, loss of follow-ups were also conducted according to the Cochrane Handbook for Systematic Review of Interventions. Meta-analysis was performed using RevMan5. 2 Software provided by Cochrane Collaboration.

RESULTSEighty-seven RCTs (involving 8 468 patients) were included. All these studies were published in Chinese. Of these only two papers were of high quality. Methods of randomization, scheme concealment, blindness, dropping-out, loss of follow-up, samples estimation were not accurately reported in the rest RCTs. The pooled results of WBC count showed that chemotherapy combined with TCHM was generally better than chemotherapy alone [MD =0. 64 x 109/L (0.41, 0.88), P < 0.01]. Auxiliary treatment of Compound Ejiao Syrup, Diyu Shengbai Tablet, Chinese compounds for invigorating Pi and supplementing Shen during the chemotherapeutic course could elevate peripheral blood WBC counts, and decrease the incidence of leucopenia.

CONCLUSIONChinese herbal medicine might have potential effects in preventing the occurrence of leucopenia, which need to be confirmed by launching higher quality clinical trials.

Drugs, Chinese Herbal ; therapeutic use ; Humans ; Leukopenia ; chemically induced ; drug therapy ; Medicine, Chinese Traditional ; Neoplasms ; drug therapy ; Phytotherapy ; Randomized Controlled Trials as Topic

Background Proliferation of the human Tenon capsule fibroblasts(HTFs) is a main cause of failure of filtering surgery.To search the drug of inhibiting the growth of the HTFs is essential for the improvement of successful rate of filtering surgery.Objective The aim of this study was to investigate the inhibitory effect of apigenin on HTFs and its mechanism.Methods Human Tenon capsular tissue was obtained during the strabismus correction surgery.HTFs was primarily cultured using explant method and identified using vimentin by immunochemistry.The 3-5 generation of cells were incubated to 96-well plate.Apigenin of 0,20,40,80,160 μmol/L was added into the medium,respectively,for 24,48,72 hours,and the proliferation of HTFs was detected by sulfonyl chloride (SRB) at the wavelength of 560 nm (A560).Bromodeoxyuridine (BrdU) of 10 μg/L was added to culture the cells for 48 hours to calculate the labeling rate of BrdU.The morphology of the cells was observed using Hoechst 33258 staining,and apoptosis and cells cycle were evaluated by flow cytometry.Results Cultured cells grew well with the positive response for vimentin,showing the green fluorescence in cytoplasm.SRB assay showed that the A560 value was gradually declined with the increase of the dosage of apigenin and prolong of time (Fgroup =480.306,P =0.000 ; Ftime =555.144,P =0.000).The labeling rate after 0,40,80 μmol/L apigenin acted for 48 hours was (87.860 ±0.632)％,(61.520±4.306)％ and (23.480±4.472)％,showing a significant difference among the three groups (F =299.347,P =0.000).The labeling rate of HTFs for BrdU was significantly decreased in the 40 and 80 μmol/L apigenin groups compared with the 0 μmol/L apigenin group (P＜0.05).Hoechse 33258 staining found that the number of the HTFs was gradually decreased and the cell number of karyopyknosis and nuclear deformation was increased with the increase of apigenin dosage.Percentage of cells in G0/G1 phase were raised and that in S and G2/M phase were declined in the higher dosage apigenin group,with a significant difference among the different groups (FG0/G1 =58.621,P=0.000;Fs =32.357,P=0.001 ;FG2/M =83.998,P=0.000).In the 72nd hour after acted by 0,40,80,160 μmol/L apigenin,the apoptosis rate of HTFs was (4.77±0.21) ％,(13.24±1.35)％,(18.33±1.86) ％,(31.58 ± 2.77) ％,respectively,with a statistically significant difference among the four groups (F =204.791,P＜0.05).Conclusions Apigenin restrains the growth of HTFs by evoking G0/G1 cell cycle arrest and inducing apoptosis in a dosage-and time-dependent manner.

Cell Proliferation ; Colony-Forming Units Assay ; Glioma ; pathology ; Hematopoietic Stem Cells ; pathology ; Humans ; Stem Cells ; cytology ; metabolism

A comprehensive analytical method based on liquid chromatography tandem mass spectrometry has been developed for the determination of nonylphenol, octylphenol and bisphenol A in textiles and food packaging) materials. Various textile and food packaging material samples were extracted under the conditions of 10.3 MPa and 120 ℃ by accelerated solvent extraction method with two static cycles using ethanol as the extraction) solvent. The extract was cleaned up by Supelclean Envi-Carb solid phase extraction cartridge. Qualitative) and quantitative analyses were carried out for the analytes under the multiple reaction monitoring(MRM) mode after the chromatographic separation on Waters XBridge C_(18))(150 mm×2.1 mm, 3.5 μm) column) with methanol-0.1% NH_4OH gradient elution. The limits of detection(LODs) for alkylphenol, octyphenol and bisphenol A were 0.5 μg/kg. The mean recoveries for textile samples at the spiked level of 0.5-10 μg/kg were 86.9%-92.5%, with the relative standard deviation less than 9.1%. The mean recoveries for food packaging material samples at the spiked level of 0.5-10 μg/kg were 87.8%-93.0%, with the relative standard deviation less than 8.8%. The method is accurate, simple, rapid, and adapts to the inspection of nonylphenol, octylphenol and bisphenol A in textiles and food packaging materials.

OBJECTIVETo observe the effect of emodin on motility signal transduction and calcium ion in colonic smooth muscle cells (SMC) in rats with bacterial peritonitis caused multiple organ dysfunction syndrome (MODS).

METHODSObservation was conducted in colon of MODS model rats on (1) effects of emodin on the contraction of muscular strip and cells of colonic smooth muscle, and influences of specific myoglobulin light chain kinase inhibitor (ML-7) and selective proteinkinase C inhibitor (Calphostin C) on these effects; and (2) effect of emodin on calcium ion in SMC.

RESULTSEmodin could directly contract the muscular strip and cells of smooth muscle; ML-7 and Calphostine could inhibit these contractile action to some extent. Under MODS condition, emodin could still increase the intracellular calcium ion concentration; this effect could be inhibited by heparin (inosamine triphosphate receptor inhibitor IP3 and ryanodine receptor inhibitor in MODS model but the calcium chelator EGTA and nifedipine (the specific cell membrane voltage dependent calcium channel blocker) showed no influence on it.

CONCLUSIONEmodin could directly contract the colonic smooth muscle in MODS model rats, which is mediated by raise the signal path MLCK of calcium ion and the PKCa path for increase calcium sensibility. The mechanism of increasing calcium ion is mainly through IP3 and RyR the two calcium ion channel receptor in the sarcoplasm.

Animals ; Calcium ; metabolism ; Colon ; drug effects ; Emodin ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Gastrointestinal Motility ; drug effects ; In Vitro Techniques ; Male ; Multiple Organ Failure ; physiopathology ; Muscle Contraction ; drug effects ; Muscle, Smooth ; cytology ; drug effects ; Myocytes, Smooth Muscle ; drug effects ; physiology ; Rats ; Rats, Wistar ; Signal Transduction

Ischemic stroke has the characteristics of high morbidity and high mortality, which seriously endanger human health.According to the statistics, ischemic stroke in China accounts for about 80% of stroke cases, and its mortality rate is as high as 50%, but most of the ischemic stroke patients who survive have sequelae, such as hemiplegia, aphasia and so on.In recent years, people have continuously studied the pathological cascade of ischemic stroke, and also achieved some good results in animal model experiments, however, the clinical results are not satisfactory. Nowadays, the better clinically effective therapeutic drug is tissue plasminogen activator, but due to the limited time window,only a small number of patients can apply this drug in time to achieve the effect.Therefore,it is essential to study drugs that are definitive and can benefit a large number of patients with ischemic stroke.The development of traditional medicines is not as fast as the development of new drugs. For ischemic stroke,we can also turn from the study of the pathogenesis of ischemic stroke to the study of traditional drugs for the endogenous neuroprotection of ischemic stroke, and look for effective targets for neuroprotection based on traditional medicine.The multi-targets and multi-effects for the acute and convalescent phases of ischemic stroke is a direction explored by researchers, which also provides some evidence for more effective drugs for the treatment of ischemic stroke.