Antigen Rv2628 of Mycobacterium tuberculosis is associated with latent tuberculosis infection. In this study, Rv2628 was prokaryotic expressed and purified, its immunological characteristics was evaluated with macrophage cell line RAW264.7 and BALB/c mice. The results show that Rv2628 was mainly expressed in form of inclusion body confirmed by SDS-PAGE, and could react with rabbit anti-H37Rv polyclonal antibody detected by Western blotting assay, indicating that the protein had an effective immunoreactivity. The interactions between Rv2628 and macrophage cell line RAW264.7 confirmed that it could effectively induce cells to produce pro-inflammatory cytokines, the relative expression level of IL-6 mRNA was higher than the control group in 1-12 h. BALB/c mice were subcutaneously immunized with Rv2628 protein, the production of IFN-gamma and IL-4 in the spleen cells was determined by Sandwich ELISA, in the Rv2628 immunized group, the level of IFN-gamma was significantly higher than that of IL-4 (P < 0.000 1). It indicated the protein induced Th1-tendency immune responses. At the same time, Rv2628(11-30) peptide used as coating antigen, the murine serum antibody titer detected by indirect-ELISA was 1:1 600, which demonstrated that Rv2628 could also induce humoral immune responses. In summary, Rv2628 could induce specific pro-inflammatory cytokines, affectively induce strongly Th1-tendency immune response and humoral response, it could be a potential target for developing subunit vaccine against TB. In addition, it laid foundation for probing the cross-talk between M. tb and host.
Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; immunology ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Interferon-gamma ; immunology ; Interleukin-4 ; immunology ; Interleukin-6 ; immunology ; Macrophages ; immunology ; Mice ; Mice, Inbred BALB C ; Mycobacterium tuberculosis ; Th1 Cells ; immunology ; Tuberculosis ; immunology

Objective To observe the effects of mind-regulating brain-developing acupoint magnetic therapy in patients with mild cogni-tive impairment (MCI). Methods From May to October, 2015, 60 patients with MCI were randomly divided into control group (n=30) and observation group (n=30). The control group received magnetic therapy on Yangming meridians, and the observation group received magnet-ic therapy on mind-regulating brain-developing acupoints, for two months. They were assessed with Montreal Cognitive Assessment (Mo-CA), digit-span and digit symbol of Wechsler Adult Intelligence Scale (WAIS). Results After treatment, the scores of MoCA, digit-span and digit symbol of WAIS significantly increased in the observation group (t>4.858, P<0.001), and were higher than that in the control group (t>2.188, P<0.05), however, only the scores of MoCA and digit symbol of WAIS significantly increased in the control group (t>5.527, P<0.001). The scores of visual space and execution, and delayed recall of MoCA increased in the observation group (t>2.324, P<0.05), and were higher than that in the control group (t>2.262, P<0.05) after treatment, and no significant difference was found in other domains (P>0.05). Conclusion Magnetic therapy on acupoints related with cognition could improve the cognitive function in patients with MCI.

Objective To explore the effect of functional electrical stimulation combined with treadmill training and botulinum toxin type A injection on foot-drop and strephenopodia among stroke survivors.Methods Sixty-seven stroke survivors with foot-drop and strephenopodia were randomly divided into an electrical stimulation group (n=23),a conventional treatment group (n =22) and a combined treatment group (n =22).All 3 groups received a 400 U injection of BTX-A and electrical stimulation.After 24 hours,the patients in the conventional treatment group received conventional treatment including a brain protection agent,limb function exercises,gait training,balance training and training in the activities of daily living.The patients in the combined treatment group received that conventional treatment,plus functional electrical stimulation and weight loss training on a treadmill.The patients in the electrical stimulation group received functional electrical stimulation treatment supplementing the conventional treatment.Therapeutic effects were evaluated before and after six weeks of treatment using integral electromyography (iEMS) of the anterior tibial muscle and the lateral head of the gastrocnemius muscle,the co-contraction ratio (CR) during ankle dorsiflexion,the modified Ashworth Scale (MAS),the Berg balance scale (BBS),a functional walking score (FAC),and the active range of motion (AROM) of the ankle in dorsiflexion and eversion.Results After the treatment,significant improvement was observed in all three groups in the average iEMS value of the anterior tibial muscle and the lateral head of the gastrocnemius muscle,the CR in ankle dorsiflexion,and in their MAS,BBS,FAC and AROM results.There was no significant difference among the three groups after treatment in their average iEMS values at the lateral head of the gastrocnemius.The average values of the other indicators were,however,significantly better in the combined treatment group than in the other 2 groups.Conclusion Functional electrical stimulation combined with treadmill training and botulinum toxin type A injection can significantly improve foot-drop,strephenopodia and the walking function of stroke survivors.This combined treatment deserves popularization and application in clinical practice.

Objective To establish a mouse model of bacterial vaginosis ( BV) by infecting estro-gen-treated mice with Prevotella bivia ( P. bivia) . Methods The mice were intraperitoneally injected with beta-estradiol 17 valerate which was suspended in sesame oil and then inoculated with different doses of P. bivia strains at the logarithmic phase. Samples of vaginal flushing fluid were collected at different time points after inoculation and used for the isolation of P. bivia strains and the detection of sialidase activities. Altogether 30 mice treated with estrogen and high dose of P. bivia were killed on days 2, 7 and 21 (n=10). Samples of cornua uteri, bladder and kidney were collected from those mice for P. bivia strains isolation. Re-sults Injection the estrogen-treated mice with P. bivia via vagina could cause the P. bivia infection for more than 14 days. The numbers of P. bivia strains isolated on day 21 decreased significantly. Enhanced sialidase activities and clue cells were observed in vaginal secretions of mice with P. bivia infection. Injection of mice with the high dose of P. bivia could spread the infection to cornua uteri. Conclusion Estrogen-treated mice could be used as an animal model for researches on BV.

Objective To evaluate the clinical performance of AQT90 FLEX,a novel time-resolved fluorescence based point-of-care test (POCT) for quantification of D-dimer in elderly patients.Methods The method from Quantitative D-dimer assay (WS/T 477-2015) for testing equipment performance was used as a reference to evaluate the clinical performance of AQT90 FLEX.The correlation was compared between testing results of D-dimer using the AQT90 immune-assay analyzer and those using the ACL TOP coagulation analyzer.Results At high concentration of D-dimer,the within-run precision coefficient of variation (CV)was 2.619％,and at low concentration of D-dimer,the within-run precision CV was 2.767％.The pollution-carrying rate was 0.12％.The measured data from AQT90 and ACL TOP had a correlation coefficient of r =0.9491 (P ＜ 0.01).The equation of the line of best fit for D-dimer with which all AQT90 results can be adapted to the ACL TOP was:AQT90 =2.52 ACL TOP + 0.15.The number from the equation was slightly greater in female than that in male,and it was also increased in elderly.Conclusions The AQT90 FLEX had rational precision and linearity in determination of concentration.There was a high agreement between the testing results from AQT90 and those from ACL TOP.It was recommended to use a slope of 2.52 and an intercept of 0.15 to adjust the D-dimer values of the ACL TOP to the AQT90 FLEX assay systems.POCT for D-dimer by AQT90 FLEX raises feasibility for use in elderly patients.

Objective To develop a multiplex allele-specific PCR (AS-PCR) assay with three-color fluo-rescence labeling for mitochondrial DNA (mtDNA) SNP typing. Methods Based on the principle of AS-PCR, the primer sets were designed for 20 SNP located on the coding region of mtDNA and divided in-to 2 groups labeled with FAM and HEX fluorescence, respectively. A primer set included two forward (reverse) allelic specific primers with different sizes and a generic reverse (forward) primer. Blood sam-ples from 200 unrelated individuals were analyzed by AS-PCR and capillary electrophoresis. Three ran-dom samples at least for each SNP site were examined and verified by direct sequencing. The haplotype frequency was investigated. Results Distinct electropherograms of 200 blood samples were obtained suc-cessfully. The typing results of direct sequencing were identical to those obtained from AS-PCR. The minimum detectable DNA concentration was 0.2 pg under the system of 10μL. The sensitivity of the DNA concentrations ranged from 0.5 to 5 pg. The 200 individuals were assigned into 15 haplotype, and the haplotype diversity was 0.906 0. Conclusion AS-PCR is a simple, rapid and efficient method for mtDNA SNP typing, and can be applied to forensic practice.

Diabetic retinopathy is one of the common and serious microvascular complications of diabetes. In foreign countries, DR is the leading cause of blindness in the working age group (20 - 64 years). In China, the incidence of DR and the rate of blindness increase year by year, which seriously affects the patients' quality of life. Previous studies on the pathogenesis and treatment of diabetic retinopathy were mainly focused on the microvascular; in recent years, with the deepening of researches, more and more scholars believe that DR is no longer simply a kind of microangiopathy, but is also accompanied by retinal neurodegeneration. However, studies on the pathogenesis of microvascular disease and neurodegenerative changes of diabetic retinopathy in the literature domestic and abroad are mostly single. This article reviews the relationship between microvascular disease and neurodegenerative changes in diabetic retinopathy.

Objective To investigate the renal expression changes of microRNA-215(miR-215) and its role in diabetic nephmpathy of type 2 diabetic db/db mice. Methods Fourweek-old diabetic db/db mice and norml control group non-diabetic db/m mice were selected.Real-time PCR was used to detect the relative level of miR-215 at the age of 8,12 and 16 weeks.Catenin beta interacting protein 1 (CTNNBIP1) mRNA and protein level were measured by realtime PCR,WesteRN blotting and immunohistochemisty.A lueiferase reporter assay was used to determine whether CTNNBIP1 was a direct target of miR-215. Results (1)With the growth of db/db mice,the major pathological characteristics of kidney included glomerular hypertrophy,segmental mesangial cells proliferation and mesangial matrix expansion.(2)Compared with the db/m mice,the db/db mice of 8,12 and 16 weeks showed obvious increase in body weight(BW),blood glucose (Glu) and 24 hour urinary albumin excretion (UAE) (P＜0.05,respectively).(3)Compared with the db/m mice,special miR-215 was highly expressed in the kidney of db/db mice and was up-regulated significantly according to the development of DN (P＜0.05).(4)The mRNA and protein expression of CTNNBIPl of kidney were consistently down-regulated in db/db mice than those in controls (P＜0.05,respectively). (5)By luciferase reporter,miR-215 could negatively regulate CTNNBIP1 gene by targeting its 3'-UTR sequence (P＜0.01). Conclusion High expression level of miR-215 plays a potential role in the initiation and progression of DN by down-regulating the expression of CTNNBIPl.

OBJECTIVE: To develop a multiplex allele-specific PCR (AS-PCR) assay with three-color fluorescence labeling for mitochondrial DNA (mtDNA) SNP typing. METHODS: Based on the principle of AS-PCR, the primer sets were designed for 20 SNP located on the coding region of mtDNA and divided into 2 groups labeled with FAM and HEX fluorescence, respectively. A primer set included two forward (reverse) allelic specific primers with different sizes and a generic reverse (forward) primer. Blood samples from 200 unrelated individuals were analyzed by AS-PCR and capillary electrophoresis. Three random samples at least for each SNP site were examined and verified by direct sequencing. The haplotype frequency was investigated. RESULTS: Distinct electropherograms of 200 blood samples were obtained successfully. The typing results of direct sequencing were identical to those obtained from AS-PCR. The minimum detectable DNA concentration was 0.2 pg under the system of 10 microL. The sensitivity of the DNA concentrations ranged from 0.5 to 5 pg. The 200 individuals were assigned into 15 haplotype, and the haplotype diversity was 0.906 0. CONCLUSION AS-PCR is a simple, rapid and efficient method for mtDNA SNP typing, and can be applied to forensic practice.
Alleles ; DNA ; DNA Primers ; DNA, Mitochondrial/analysis* ; Electrophoresis, Capillary ; Haplotypes ; Humans ; Mitochondria ; Polymerase Chain Reaction/methods* ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA

This study was aimed to investigate the biological characteristics of osteoblasts from patients with myelodysplastic syndrome (MDS) and their supportive capacity for hematopoiesis in vitro. A two-dimensional culture system was constructed by using osteoblasts derived from human marrow mesenchymal stem cells (MSC); MSCs were isolated from bone marrow of MDS patients and normal individuals and were cultured; the third passage of MSCs were induced into osteoblasts which were treated with mitomycin C and confluenced into a feeder layer. Ficolled bone marrow mononuclear cells were obtained from normal individuals and seeded into the two-dimensional culture system to culture in vitro without exogenous cytokines. By using colony-forming assay, the ability of the two-dimensional system to culture HPCs was observed. The cytokine expression of osteoblasts from MDS patient bone marrows in mRNA level was detected by RT-PCR and was compared with human osteoblast cell line hFOB1.19. The results showed that the osteoblasts from MDS patients could support short-term survival of GM-CFC in condition without exogenous cytokines, that is, osteoblasts played a crucial role in regulation of HPC growth. The results of RT-PCR clearly demonstrated that the osteoblast cell line hFOB1.19 expressed SCF, IL-6, SDF-1alpha, G-CSF and GM-CSF. The same expression patterns of above cytokines were also seen in osteoblasts derived from BM-MSCs of MDS patients and normal individuals, but these cells did not express GM-CSF. It is concluded that the biological characteristics of osteoblasts from bone marrow of MDS patients are generally not different from those of osteoblasts from normal bone marrow. Both of them can support GM -CFC to form colonies in vitro, it may be associated with expressing important related cytokines by osteoblasts.
Cytokines ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Interleukin-6 ; metabolism ; Myelodysplastic Syndromes ; metabolism ; pathology ; Osteoblasts ; metabolism ; physiology ; RNA, Messenger ; metabolism ; Stem Cell Factor ; metabolism