Full-length NS5A gene of the hepatitis C virus was amplified by PCR using plasmid pBAC25 containing HCV nonstructural gene as template. The amplified fragment (about 1.34 kb) was cloned into plasmid pQE32, and the recombinant plasmid pQENS5A was expressed in JM109 strain. The NS5A protein was purified by NiSO4 metal chelating resin, and characterized by Western-blot. Its antigenecity was determined by ELISA. The positive detection rate of anti-NS5A was 75% (69/92) in ninety-two clinic sera. The positive rate of anti-NS5A was 82.5% (33/40) in fourty positive standand sera, and the negative rate of anti-NS5A was 100% (40/40) in fourty negative standand sera. The results showed that the Full-length NS5A proteinn had the higher sensitivity and specificity in the detection of HCV antibody in sera, we suggested that NS5A protein was a useful antigen for blood screening.

OBJECTIVETo evaluate the clinical efficacy of intradermal injection of methylene blue for treatment of moderate to severe acute thoracic herpes zoster and prevention of postherpetica neuralgia in elderly patients.

METHODSSixty-four elderly patients with herpes zoster were randomized to receive a 10-day course of intradermal injection of methylene blue and lidocaine plus oral valaciclovir (group A, 32 cases) and intradermal injection of lidocaine plus oral valaciclovir (group B).Herpes evaluation index, pain rating index, incidence of postherpetic neuralgia, and comprehensive therapeutic effect were compared between the two groups at 11, 30 and 60 days after the treatment.

RESULTSThe baseline characteristics were comparable between the two groups (all P>0.05). Compared with that in group B, the time for no new blister formation, blister incrustation and decrustation, and pain relief was significantly shortened in group A (P<0.05) with also obviously lower pain intensity after the treatment. The incidence of postherpetic neuralgia was significantly lower in group A than in group B at 30 days (P<0.05), but not at 60 and 90 days after the treatment. The total clinical response rate was 93.8% in group A, much higher than that in group B (62.5%, P<0.05).

CONCLUSIONIntradermal injection of methylene blue can effectively shorten the disease course, reduce the pain intensity and prevent the development of postherpetic neuralgia in elderly patients with herpes zoster.

Acyclovir ; administration & dosage ; analogs & derivatives ; therapeutic use ; Aged ; Herpes Zoster ; complications ; Humans ; Incidence ; Injections, Intradermal ; Lidocaine ; administration & dosage ; therapeutic use ; Methylene Blue ; administration & dosage ; therapeutic use ; Neuralgia, Postherpetic ; therapy ; Pain Measurement ; Valine ; administration & dosage ; analogs & derivatives ; therapeutic use

OBJECTIVETo construct a GFP/Puro double-labeled lentiviral expression vector for CK8 silencing and assess the effects of CK8 silencing on cell apoptosis.

METHODSThe siRNA sequences of CK8 were inserted into the lentiviral expression vector GV248 and transfected into 293T cells with the packaging plasmids PMD and SPA. The lentivirus was collected at 24 and 36 h post-transfection. Flow cytometry was used to detect the virus titer and the positive cells were selected with puromycin. The knockdown of CK8 was examined by Western blotting. The effect of CK8 down-regulation on cell apoptosis induced by cisplatin was detected with Annexin V/PI staining.

RESULTS AND CONCLUSIONWe successfully constructed CK8 interference lentiviral vector and obtained a stable cell line with CK8 knock-down that was sensitive to cisplatin-induced apoptosis.

Apoptosis ; Cell Line ; Down-Regulation ; Genetic Vectors ; Humans ; Keratin-8 ; genetics ; Lentivirus ; Plasmids ; RNA Interference ; RNA, Small Interfering ; Transfection

To study the effects of supernatant derived from acute myeloid leukemia (AML) cell lines on proliferation and apoptosis of CD4(+) and CD8(+) T cell subsets and to investigate the mechanism by which AML escapes from immune recognition, lymphocytes were labeled with CFSE and were stimulated with anti-CD3 and anti-CD28 in presence or absence of supernatants from three AML cell lines (HL-60, NB4, U937). After culture, cell suspensions were labeled with 7AAD and CD4 PE (or CD8 PE). Cells were then detected by flow cytometry and their proliferation and apoptosis were analyzed. The results showed that supernatants from two of three cell lines (HL-60 and NB4) inhibited the proliferation of CD4(+) and CD8(+) T cells, and the degree of inhibition showed a dose-dependent way. Similarly, the apoptosis of stimulated CD4(+) T cells was inhibited, but stimulated CD8(+) T cells remained unaffected by supernatant from HL-60 and NB4. In contrary, the apoptosis of proliferative CD8(+) T cells were increased significantly by HL-60 and NB4 supernatant. It is concluded that soluble factors derived from AML cell lines inhibit the proliferation of CD4(+) and CD8(+) T cells and induce the apoptosis of proliferative CD8(+) T cells, that may be one of the mechanisms by which the immunity was suppressed.
Apoptosis ; physiology ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Cells, Cultured ; Culture Media ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; immunology ; pathology ; T-Lymphocytes ; cytology ; Tumor Cells, Cultured ; U937 Cells

OBJECTIVETo investigate the effects of inhibiting TIM-4 function in Kupffer cells (KCs) on liver graft rejection in mice and explore the underlying mechanism.

METHODSMouse models of orthotopic liver transplantation were treated with a control mAb group and TIM-4 mAb. The activated KCs were assayed with immunohistochemistry after operation. The expression of TIM-4 in KCs were assayed with Western blotting and RT-PCR and the levels of AST, ALT, TBIL, TNF-α, IFN-γ and CCL2 were assayed detected. The expression of TIM-4 in KCs was observed with laser confocal microscopy. HE staining was used to observe the microstructure of the liver tissues, and the number of CD25Foxp3T cells was determined using with flow cytometry; the proteins levels of p-P65and p-P38 were assayed with Western blotting. The donor mice were treated with clodronate liposomes to destroy the KCs in the liver before transplantation, and the liver grafts were examined for graft rejection.

RESULTSThe number of activated KCs in the liver graft increased progressively over time. Compared with the sham-operated group, the liver graft showed significantly increased TIM-4 protein and mRNA levels at 1, 3, and 7 days after transplantation (P<0.05) and increased levels of AST, ALT, TBIL, TNF-α, IFN-γ and CCL2 at 7 days (P<0.05). The graft in TIM-4 mAb group showed mild pathological changes with a mean RAI score of 2.67∓0.75, which was significantly lower than that in control mAb group (P<0.05). The mean survival time of the recipient mice was 53.8∓6.4 days in TIM-4 mAb group, significantly longer than that in the control mAB group (14.5∓2.9 days, P<0.05). Donor treatment with clodronate liposomes resulted in comparable RAI scores in TIM-4 mAb and control mAb groups (8.01∓0.64 vs 7.93∓0.56, P>0.05). The protein levels of p-P65 and p-P38 in TIM-4 mAb group were significantly lower than those in control mAb group (P<0.05), and CD25Foxp3T cells in the liver graft increased significantly in TIM-4 mAb group.

CONCLUSIONInhibition of TIM-4 function in KCs reduces the production of inflammatory factors after liver transplantation possibly by inhibiting the NF-κB and MAPK signaling pathways and promoting the proliferation of Foxp3Treg cells to induce allograft tolerance.

Animals ; Antibodies, Monoclonal ; pharmacology ; Graft Rejection ; Immunohistochemistry ; Kupffer Cells ; drug effects ; metabolism ; Liver ; surgery ; Liver Transplantation ; Male ; Membrane Proteins ; antagonists & inhibitors ; Mice ; NF-kappa B ; metabolism ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo investigate the inhibitory effects of apatinib on colorectal carcinoma HCT-116 cells in vitro and the signaling pathways involved.

METHODSThe cytotoxicity of different concentrations (0, 0.5, 1, 1.5, and 2 µmol/L) of apatinib in HCT-116 cells was assessed by MTT assay, using capecitabine as the positive control. The apoptosis rate of apatinib-treated HCT-116 cells was detected using flow cytometry, and the expressions of Bcl-2, Bax, and caspase-3 were determined with quantitative real-time PCR and Western blotting. The effect of apatinib on the expressions of Akt, pAkt, Erk1/2 and pErk1/2 in HCT-116 cells was evaluated using Western blotting.

RESULTSApatinib significantly inhibited the proliferation of HCT-116 cells in a concentration-dependent manner with an ICvalue of 1.335 µmol/L. Flow cytometric analysis showed that apatinib significantly increased the apoptotic rate of HCT-116 cells dose-dependently. Apatinib induced the expression of the pro-apoptotic genes Bax and caspase-3 at both the mRNA and protein levels while inhibited the expression of the anti- apoptotic gene Bcl-2. The expressions of p-Akt and p-Erk1/2 were decreased in HCT-116 cells after apatinib treatment, but the total protein levels did not undergo obvious changes.

CONCLUSIONApatinib inhibits the proliferation and induces apoptosis of HCT-116 cells by suppressing the phosphorylation of Erk1/2 and Akt in the MAPK/Erk and PI3K/Akt signaling pathways.

OBJECTIVETo investigate a new strategy of polylactic acid (PLA) nanoparticles delivery system coating nuclear factor-kappaB (NF-kappaB) decoy oligonucleotides (ODNs) for inhibiting TF expression in cultured brain microvascular endothelial cells(BMECs).

METHODSPLA nanoparticles coating FITC-labeled NF-kappaB decoy ODNs were formulated by nano-deposition method and the characteristics of nanoparticles were detected. BMECs were isolated and cultured in vitro. The cellular uptake and intracellular localization of nanoparticles in BMECs was detected by flow cytometry and confocal microscopy. Changes in the expressions of TF and nuclear protein P65 were examined by RT-PCR and Western blot in NF-kappaB decoy ODNs transfected BMECs by LPS stimulation.

RESULTSThe decoy-nanoparticles obtained were uniform spherical particles with an effective diameter of 162.1 nm and a polydispersity index of 0.118. NF-kappaB decoy ODNs encapsulated in nanoparticles could be released in a controlled manner in phosphate-buffered saline for up to 28 days. It was observed that the cellular uptake of nanoparticles were increased with the time of incubation and the concentration of nanoparticles in the medium. Nanoparticles localized mainly in the BMECs cytoplasm. LPS-induced upregulation of TF transcription was inhibited by NF-kappaB decoy ODNs transfection but not by missense ODNs transfection. Furthermore, changes in the transcription level of TF were paralleled by a reduction of capacity of P65 in nuclear extract of NF-kappaB decoy ODNs transfected cells.

CONCLUSIONSThese findings offer a potential therapeutic strategy in the control of TF expression in BMECs in vitro.

Animals ; Brain ; blood supply ; Capillaries ; cytology ; Cells, Cultured ; Endothelial Cells ; metabolism ; Gene Expression Regulation ; Lactic Acid ; NF-kappa B ; genetics ; Nanoparticles ; Oligonucleotides ; genetics ; Polyesters ; Polymers ; Rats ; Thromboplastin ; genetics ; metabolism ; Transfection

OBJECTIVETo cultivate hematopoietic stem/progenitor cells (CD34(+)CD38(-)) isolated from umbilical cord blood (UCB) long for the observation of cell growth and expansion in vitro, surface marker expression, and chromosomal complements.

METHODSBy flow cytometry CD34-FITC and CD38-PE labeled CD34(+) and CD38(-) stem/progenitor cells were isolated from UCB. The cells were cultivated in vitro for 6 months in a stem cell culture system with addition of six kinds of cell growth factors (IL-3, IL-6, GM-CSF, Epo, SCF, IGF-1). One month after cultivation, cultured cells were investigated for surface marker expression by flow cytometry and karyotype by G banding method.

RESULTSAfter 7-12 days cultivation, the CD34(+)CD38(-) stem/progenitor cells began proliferation. The proliferation rate and the peak proliferation duration were greater in 1 cell/well cultivation conditions than in 10 cells/well. The cells remained CD34(+)CD38(-) and their karyotypic characteristics remained unchanged.

CONCLUSIONCD34(+)CD38(-) stem/progenitor cells from UCB may provide a larger than original amount of stem/progenitor cells for transplantation after long-term cultivation in vitro.

ADP-ribosyl Cyclase 1 ; immunology ; Adult ; Antigens, CD34 ; immunology ; Cell Proliferation ; Cells, Cultured ; Female ; Fetal Blood ; cytology ; immunology ; Hematopoietic Stem Cells ; cytology ; immunology ; Humans ; Immunophenotyping ; Karyotyping

OBJECTIVETo investigate the expression of long non-coding RNA HOTAIR in the plasma of breast cancer patients and its value in the diagnosis of breast cancer.

METHODSHOTAIR levels were measured in 24 tumor tissues and 70 plasma samples from breast cancer patients using quantitative real-time PCR. The correlations of plasma HOTAIR level with the clinicopathological features of the patients were analyzed. A multivariate logistic regression model was established to analyze the value of plasma HOTAIR in comparison with plasma CA153 and CEA levels for breast cancer diagnosis. We further detected HOTAIR levels in the plasma and breast cancer tissues of 24 patients before and after operation and investigated their correlation.

RESULTSBreast cancer patients had increased expressions of HOTAIR in the tumor tissues and plasma, and plasma HOTAIR level was significantly correlated with estrogen receptor (ER) level (P=0.004) and lymph node metastasis (P=0.010). Receiver operating characteristic (ROC) curve and the multivariable logistic regression model showed that the area under ROC curve (AUC) of plasma HOTAIR was 0.82 (P<0.001) for breast cancer diagnosis with a diagnostic sensitivity and a specificity of 73.3% and 93.3%, respectively. The diagnostic power and specificity of plasma HOTAIR was much higher than those of CA153 (AUC=0.66, P=0.030) and CEA (AUC=0.52, P=0.001), and the combination of the 3 markers further enhanced the diagnostic power (AUC=0.84) and specificity (96.7%). Plasma HOTAIR level was significantly reduced in the patients after the operation (P<0.0001) and showed a moderate correlation with its expression in tumor tissues (r=0.62, P<0.0001).

CONCLUSIONPlasma HOTAIR may serve as a potential biomarker for breast cancer diagnosis.

Biomarkers, Tumor ; blood ; Breast Neoplasms ; blood ; diagnosis ; Carcinoembryonic Antigen ; blood ; Female ; Humans ; Logistic Models ; Lymphatic Metastasis ; Mucin-1 ; blood ; Prognosis ; RNA, Long Noncoding ; blood ; ROC Curve ; Real-Time Polymerase Chain Reaction ; Receptors, Estrogen ; metabolism ; Sensitivity and Specificity

OBJECTIVETo distinguish the edema, injury, or rupture in the traumatic skeletal muscle fiber in vivo using diffusion tensor imaging (DTI) and tractography on magnetic resonance imaging (MRI).

METHODSThe skeletal muscle trauma models were made in 4 rabbits (eight hindlimbs) by iron discus (weight 1.0 kg, diameter 6 cm) falling down vertically from 45 cm height to rabbits' thighs. Conventional sequences and two-dimensional (2D) diffusion-weighted (DW) spin-echo (SE) echo planar imaging (EPI) sequence with fat suppression (b = 600 s/mm2) were performed on 1. 5T MRI scanner. The grading of edema, injury, and fiber rupture in the damaged muscle were made according to their histopathological views, which was consistent with the images. The mean apparent diffusion coefficient (ADC) values and fractional anisotropy (FA) values were measured from the region of interests (ROIs) of all groups on 2D DW images used for tractography. Analysis of variance test was performed to analyze all data.

RESULTSADC values of the areas in normal muscle, edema muscle, injury muscle, and ruptured muscle were (6.12 +/- 1.34) x 10(-3), (6.38 +/- 1.30) x 10(-3), (8.06 +/- 0.97) x 10(-3), and (9.57 +/- 0.93) x 10(-3) mm2/s, respectively. There was significant difference among groups (P < 0.001), but no difference between edema muscle and normal muscle group (P > 0.05). The FA values of normal muscle, edema muscle, injury muscle, and ruptured muscle were 0.42 +/- 0.12, 0.36 +/- 0.12, 0.26 +/- 0.09, 0.12 +/- 0.08, respectively, with a significant difference among groups (P < 0.001). In the edema muscle, the tracking cross-fiber could be seen but it decreased slightly. In the injury muscle, the tracking fiber decreased markedly. In the ruptured muscle, the transverse-orientation tracking fiber vanished, yet some interrupted longitudinal-orientation tracking fiber could be found.

CONCLUSIONThe edema, injury, and rupture of muscle fiber in rabbit damaged skeletal muscle can be verified according to the ADC and the FA on DTI and tractography.

Animals ; Anisotropy ; Diffusion Magnetic Resonance Imaging ; methods ; Echo-Planar Imaging ; Edema ; diagnosis ; pathology ; Male ; Muscle, Skeletal ; injuries ; pathology ; Rabbits ; Rupture ; diagnosis ; pathology ; Thigh ; injuries ; pathology