Objective: To observe the clinical efficacy of acupoint injection with salmon calcitonin for back pain in elderly patients with primary osteoporosis. Methods: A total of 76 patients were selected and randomly divided into two groups by the random number table method, with 39 cases in the treatment group and 37 cases in the control group, respectively. Patients in both groups received routine anti-osteoporosis treatment. Patients in the treatment group received additional acupoint injection with salmon calcitonin at bilateral Pishu (BL 20) and Shenshu (BL 23), while patients in the control group received additional intramuscular injection with salmon calcitonin. The treatments for both groups were given once a day and lasted for 4 weeks. Visual analog scale (VAS) and Chinese Oswestry disability index (CODI) scores were observed before treatment, after 2 weeks and 4 weeks of treatment, and the use of analgesics during the treatment were recorded. Results: After treatment, the clinical efficacy in the treatment group was more significant than that in the control group (P<0.05). After 2 weeks and 4 weeks of treatment, the VAS scores in both groups showed significant intra-group and between-group differences (all P<0.05), and the CODI scores in both groups showed significant intra-group differences (all P<0.05). After 2 weeks of treatment, the CODI score showed no significant between-group difference (P>0.05). After 4 weeks of treatment, the improvement of CODI score in the treatment group was more significant than that in the control group (P<0.05). During the treatment, 2 cases in the treatment group took analgesics versus 8 cases in the control group, and the result showed a significant between-group difference (P<0.05). Conclusion: For back pain in elderly patients with primary osteoporosis, based on the routine treatment of oral medication, the clinical efficacy of acupoint injection with salmon calcitonin at bilateral Pishu (BL 20) and Shenshu (BL 23) is more significant than that of intramuscular injection. Acupoint injection treatment can improve patients' conditions and reduce the use of analgesics.

Adult ; Electromyography ; Gasoline ; poisoning ; Humans ; Male ; Poisoning ; physiopathology ; psychology ; Young Adult

Objective To investigate the effects of CD4+T cell depletion in BALB/c mice on the immunogenicity and virulence of replication-competent recombinant vaccinia virus. Methods Twelve BALB/c mice were inoculated with recombinant Tiantan vaccinia ( rTV, n=8 ) or vaccinia virus Tiantan strain ( VTT, n=4) by tail scarification after the depletion of CD4+T cells with anti-CD4 monoclonal anti-body( McAb) injected intraperitoneally for three days before virus inoculation.A control group without anti-body treatment was set up accordingly.Several parameters including the body weight, pocks on tail, viral shedding and the percentage of CD4+T cells were monitored.In the fourth week after virus infection, ovaries were collected from mice and viral loads in the tissue were titrated by plaque forming assay on chick embryo fibroblast ( CEF) cells.The specific cellular immune responses against vaccinia virus and HIV induced by inoculation of VTT and rTV respectively were detected by intracellular cytokine staining ( ICS) assay.ELISA was used to detect antibodies against vaccinia virus and HIV-1 gp120.Results All mice with or without CD4 McAb treatment showed typical poxvirus pocks on tails after inoculation of vaccinia viruses, but none of them developed secondary or satellite lesions.It took a longer time for CD4+T cell-depleted mice to heal from lesions, to regain body weights and to release viruses than the mice in control group.No vaccinia virus was detected in the ovaries of CD4+T cell-depleted mice or mice in control group.The mean absorbance( A) values for the detection of HIV-specific and vaccinia virus-specific antibodies in CD4+T cell-depleted mice with ELISA were respectively 0.119 and 0.168, which were significantly lower than those in mice of control group.The titers of neutralizing antibodies against vaccinia virus in McAb/rTV treated mice (1 ∶321) were lower than those in rTV treated mice (1 ∶1286) (P<0.05).The percentages of CD4+T cells secreting IFN-γ(0.654%) in McAb/rTV treated mice were significantly lower than those in rTV treated mice in the fourth week after immunization (P <0.0004).No significant differences with the vaccinia virus-specificCD8+ T cell responses were observed among mice with or without CD4+T cells depletion.Conclusion Thereplication and dissemination of replication-competent recombinant vaccinia virus could be effectively controlledin the mice with CD4+ T cell-depletion.The depletion of CD4+ T cells significantly reduced the humoraland CD4+ T cell responses, but had no effect on CD8+ T cell responses.

Objective To analyze the antibody responses in guinea pigs vaccinated with recombi-nant vaccinia virus( rTT) strains expressing transmitted/founder ( T/F) HIV-1 membrane proteins in combi-nation with gp140 protein.Methods Guinea pigs were primed with rTT strains and boosted twice with gp140 protein in every four weeks.Serum samples were collected from guinea pigs before immunization and in 2, 6 and 10 weeks after the last immunization for the detection of HIV-1-specific binding antibodies, neu-tralizingantibodiesandtherelativeavidityofantibodies.Results (1)Thebindingantibodiesspecificto HIV-1 B′/C, B, AE subtypes were efficiently induced by the immunization of rTT-B, rTT-C and rTT-CON vaccinia strains in combination with gp140 protein.The antibody titers ranged from 111 430 to 1 024 000. More antibodies against HIV-1 B′/C and AE subtypes were induced in guinea pigs by the immunization of rTT-C and rTT-CON strains in combination with gp140 protein than those by using rTT-B strain prime-protein boost strategy (P<0.05).No significant differences with the titers of HIV-1 B subtype specific antibody were observed among the guinea pigs immunized with the three strategies.( 2 ) High titers of SF162 and ZM109 neutralizing antibodies were induced in guinea pigs immunized with rTT-B, rTT-C and rTT-CON vac-cinia strains in combination with gp140 protein, ranging from 83.76 to 649.30.No significant differences were found among the three groups.(3) The HIV-1 V1V2-gp70 specific antibodies associated with protec-tive immunity were induced by immunization of the three virus prime-protein boost strategies.No significant differences were observed among them.(4) Antibodies induced in guinea pigs by immunization of the three strategies showed strong affinity to membrane proteins of HIV-1 B′/C, B, AE subtype strains.No significant differences were found among the three immunization strategies.Conclusion A strong humoral immune re-sponse was induced in guinea pigs primed with recombinant vaccinia virus strains expressing T/F virus HIV-1 membrane proteins and boosted with gp140 protein.

Objective To assess the prevalence and binocular dysfunctional risk factors associated with asthenopia among adult myopes.Methods The study population included 800 adult myopes,a cross-sectional visual parameters that characterize the accommodative:accommodation amplitude (AA),accommodative facility,and accommodative response (fused crossed cylinder-FCC) and binocular function (near and distant horizontal and vertical associated phorias,near and distance negative and positive fusional vergence,near point of convergence,negative and positive relative accommodation (NRA/PRA),stimulus AC/A ratio and stereoacuity) were evaluated when these subjects wore adequate spectacle correction.Results Asthenopia was reported in 24.2％ (194/800) of myopes.The incidence of asthenopia in female (27.8％,128/460) was more than that in male (19.4％,66/340),and there was significant difference (P =0.006).In univariate analysis,the monocular AA,binocular AA,NRA and PRA were significantly associated with asthenopia (P =0.000).In multivariate analysis,low NRA (≤1.25 D),low NRA (≤1.50 D) were significant risk factors for asthenopia (P =0.000,OR =7.644 ;95％ CI 2.913-17.580;P =0.000,OR =5.303;95％ CI 2.822-16.205).Conclusion Preventive measures directed against the binocular dysfunctional risks factors associated with asthenopia may help reduce the prevalence and provide a positive impact on asthenopia.

The positive ratio is 74 percent using the plate anaerobe culturing device made by author, higher than using common anaerobic box and anaerobic jar.

miR-200c has been shown to regulate the epithelial-mesenchymal transition (EMT) by inhibiting ZEB1 and ZEB2 expression in breast cancer cells. This study further examined the role of miR-200c in the invasion and metastasis of breast cancer that goes beyond the regulation on ZEB1 and ZEB2 expression. In this study, the bioinformatics software (miRanda) was used to predict the target gene of miR-200c and Renilla luciferase assay to verify the result. The metastatic breast cancer cells MDA-MB-231 were cultured and transfected with the miR-200c mimic or inhibitor. The expressions of miR-200c and HMGB1 were detected by RT-PCR and Western blotting, respectively. Transwell assay and wound healing assay were employed to examine the invasive and migrating ability of transfected cells. Target prediction and Renilla luciferase analysis revealed that HMGB1 was a putative target gene of miR-200c. After transfection of MDA-MB-231 cells with the miR-200c mimic or inhibitor, the expression of miR-200c was significantly increased or decreased when compared with cells transfected with the miR-200c mimic NC or inhibitor NC. Moreover, the expression of HMGB1 was reversely correlated with that of miR-200c in transfected cells. Tranwell assay showed that the number of invasive cells was significantly reduced in miR-200c mimic group when compared with miR-200c inhibitor group. It was also found that the migrating ability of cells transfected with miR-200c mimics was much lower than that of cells transfected with miR-200c inhibitors. It was suggested that miR-200c can suppress the invasion and migration of breast cancer cells by regulating the expression of HMGB1. miR-200c and HMGB1 may become useful biomarkers for progression of breast cancer and targets of gene therapy.

To reduce the serum clearance of interferon alpha2b, a chimeric gene encoding an human serum albumin(HSA)--human interferon alpha2b(IFNalpha2b) fusion protein was overexpressed in Pichia pastoris. After fermentation in a 5L bioreactor, the fusion protein, capable of cross-reacting with anti-IFN alpha and anti-HSA antibody, was purified from the culture of the recombinant yeast by ultrafiltration, blue Sepharose affinity, phenyl hydrophobic interaction and Q ion exchange chromatography. Its IFNa2b moiety exhibits antiviral activity similar to that of recombinant human IFNa2b. In Cynomolgus monkeys model, The fusion protein was detectable in plasma, even 336h after a single does of 90 microg/kg injection intravenously or subcutaneously. The elimination phase half-life of the fusion protein was 101h after intravenous injection and 68.2h after subcutaneous injection. Its Subcutaneous bioavailability was 67.9%. The enhanced pharmacokinetics of interferon a2b fused to human serum albumin suggest its promissing application in clinic medicine.
Animals ; Bioreactors ; microbiology ; Fermentation ; Humans ; Interferon-alpha ; biosynthesis ; genetics ; Macaca fascicularis ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacokinetics ; Recombinant Proteins ; Serum Albumin ; biosynthesis ; genetics

BACKGROUNDIn vitro chondrocyte expansion is a major challenge in cell-based therapy for human articular cartilage repair. Classical culture conditions usually use animal serum as a medium supplement, which raises a number of undesirable questions. In the present study, two kinds of defined, serum-free media were developed to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering.

METHODSBovine chondrocytes were expanded in serum-free media supplemented with fibroblast growth factor-2 and platelet-derived growth factor or fibroblast growth factor-2 and insulin-like growth factor. Expansion culture in a conventional 10% fetal bovine serum (FBS) medium served as control. Fibronectin coating was used to help cell adhesion in serum-free medium. Next, in vitro three-dimensional pellet culture was used to evaluate the chondrocyte capacity. Cell pellets were expanded in different media to re-express the differentiated phenotype (re-differentiation) and to form cartilaginous tissue. The pellets were assessed by glycosaminoglycans contents, collagen II, collagen I and collagen X immunohistological staining.

RESULTSChondrocytes cultured in serum-free media showed no proliferation difference than cells grown with 10% FBS medium. In addition, chondrocytes expanded in both serum-free media expressed more differentiated phenotypes at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I. Pellets derived from chondrocytes cultured in both serum-free media displayed comparable chondrogenic capacities to pellets from cells expanded in 10% FBS medium.

CONCLUSIONThese findings provide alternative culture approaches for chondrocytes in vitro expansion, which may benefit the clinical use of autologous chondrocytes implantation.

Animals ; Cartilage, Articular ; cytology ; Cattle ; Cell Dedifferentiation ; Cells, Cultured ; Chondrocytes ; cytology ; physiology ; Culture Media, Serum-Free ; Fibronectins ; pharmacology ; Real-Time Polymerase Chain Reaction ; SOX9 Transcription Factor ; genetics

Interleukin-2 (IL-2) was initially isolated as a T cell growth factor and had been shown to direct the expansion and differentiation of several hematopoietic cell types. Clinical studies using IL-2 in the treatment of AIDS have been encouraging, due to its critical role as a proliferative signal for activated T-lymphocytes. IL-2 has also undergone trials in the treatment of several types of cancer, based on its stimulation of cytotoxic, antitumor cells. Today, human IL-2 is produced completely by genetically engineered method, and it has been proved that genetically engineered recombinant human IL-2 has almost the same function and clinical effect as wild IL-2. In the former study, recombinant human IL-2 usually comes from E. coli, in this paper the mutant IL-2 was successfully expressed and purified in Pichia pastoris for the first time. As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression level. Expression conditions of human mutant interleukin-2(the codon for cysteine-125 of human IL-2 with alanine; the codon for leucine-18 with methionine; the codon for leucine-19 with serine) in the recombinant Pichia pastoris strain were optimized via test of some factors such as the rate of aeration, the inductive duration, the initial pH and the concentration of methanol. The results from tests showed that the most important parameter for efficient expression of interleukin-2 in recombinant Pichia pastoris strain is adequate aeration during methanol induction, and the optimum inductive condition for interleukin-2 expression was: more than 80% aeration, 2 days for induction, the initial pH of 6.0, the final methanol concentration of 1.0%. With this condition, the expressed IL-2 was secreted into fermentation broth and reached a yield of 30%, approximately 200 mg/L. Expressed interleutin-2 (MvIL-2) was isolated and purified by centrifugation, millipore filtration to concentration, Econo-PacS strongly acidic cation exchanger cartridge and molecular sieve chromatography and the yield of MvIL-2 was 27%. MvIL-2 was purified to electrophoretic purity by SDS-PAGE and only one peak being loaded on HPLC. Purified MvIL-2 protein had stimulating activity similar to the wild type of IL-2 as assayed by IL-2-dependent CTLL-2 cells. However, the stability of MvIL-2 was superior than that of IL-2 at different temperatures. The activity of obtained MvIL-2 was 4 - 5 times of the wild type of IL-2, So MvIL-2 had an advantage over wild type of rhIL-2 in storage stability and activity.
Fermentation ; Humans ; Interleukin-2 ; biosynthesis ; genetics ; Mutant Proteins ; biosynthesis ; Mutation ; Pichia ; genetics ; metabolism ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification