Urokinase plasminogen activator receptor (uPAR) is a membrane protein which is attached to the cellular external membrane. The uPAR expression can be observed both in tumor cells and in tumor-associated stromal cells. Thus, in the present study, the human amino-terminal fragment (hATF), as a targeting element to uPAR, is used to conjugate to the surface of superparamagnetic iron nanoparticle (SPIO). Flowcytometry was used to examine the uPAR expression in different tumor cell lines. The specificity of hATF-SPIO was verified by Prussian blue stain and cell phantom test. The imaging properties of hATF-SPIO were confirmed in vivo magnetic resonance imaging (MRI) of uPAR-elevated colon tumor. Finally, the distribution of hATF-SPIO in tumor tissue was confirmed by pathological staining. Results showed that the three cells in which we screened, presented different expression characteristics, i. e., Hela cells strongly expressed uPAR, HT29 cells moderately expressed uPAR, but Lovo cells didn't express uPAR. In vitro, after incubating with Hela cells, hATF-SPIO could specifically combined to and be subsequently internalized by uPAR positive cells, which could be observed via Prussian blue staining. Meanwhile T2WI signal intensity of Hela cells, after incubation with targeted probe, significantly decreased, and otherwise no obvious changes in Lovo cells both by Prussian blue staining and MRI scans. In vivo, hATF-SPIO could be systematically delivered to HT29 xenograft and accumulated in the tumor tissue which was confirmed by Prussian Blue stain compared to Lovo xenografts. Twenty-four hours after injection of targeting probe, the signal intensity of HT29 xenografts was lower than Lovo ones which was statistically significant. This targeting nanoparticles enabled not only in vitro specifically combining to uPAR positive cells but also in vivo imaging of uPAR moderately elevated colon cancer lesions.
Cell Line, Tumor ; Colonic Neoplasms ; diagnosis ; Ferric Compounds ; Humans ; Magnetic Resonance Imaging ; Magnetite Nanoparticles ; chemistry ; Molecular Imaging ; methods ; Receptors, Urokinase Plasminogen Activator ; chemistry ; Staining and Labeling

To study the regulation of androgen receptor (AR) expression in human prostate cancer LNCaP cells by triptolide (TP) and the possible mechanism, by using qRT-PCR and Western blot, the AR mRNA and protein levels in TP treated LNCaP cells were detected, and the AR protein level in TP and NF-κB inhibitor treated LNCaP cells was also detected; a series of pGL3-AR promoter reporter gene vectors were built using restriction-free cloning method, and the vectors were employed to investigate the effects of TP on the transcriptional activity of AR promoter in LNCaP cells; the upstream proteins which may play regulatory roles were detected using western blot assay. After treated LNCaP cells with TP for 48 h, AR mRNA and protein expressions decreased with increasing TP concentration. The expression of AR target gene PART1 and prostate specific antigen (PSA) was also downregulated by TP treatment; a series of pGL3-AR promoter reporter vectors were constructed and validated by sequencing and luciferase activity; the results of dual luciferase reporter assay showed that TP downregulated AR at the transcriptional level; PI3K/AKT/NF-κB pathway which is associated with AR promoter activity was drowregulated by TP. In conclusion, our results demonstrated that the transcriptional activity of AR in LNCAP cells was downregulated by TP, and PI3K/AKT/NF-κB pathway may be involved in the regulation mechanism.
Cell Line, Tumor ; Diterpenes ; pharmacology ; Down-Regulation ; Epoxy Compounds ; pharmacology ; Genetic Vectors ; Humans ; Male ; NF-kappa B ; antagonists & inhibitors ; Phenanthrenes ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Promoter Regions, Genetic ; Prostate-Specific Antigen ; metabolism ; Prostatic Neoplasms ; metabolism ; RNA, Messenger ; Receptors, Androgen ; metabolism ; Signal Transduction ; Transcriptional Activation

Objective To compare the single-shot fields irradiated by three focusing modes of γ-knife and explore the approaches for improving the quality of stereotactic radiosurgery.Methods GAFCHROMIC(R) EBT3 mode flushing-free film was used to measure the single-shot fields irradiated by multi-source static focusing modes,multi-source single-axis rotating focusing mode and single-source double-axis rotating focusing mode of γ-knife.Also the uniformity and penumbra of the single-shot fields were compared.Results The 2D dose distribution of the single-shot fields irradiated by three focusing modes of γ-knife was different.In the axis (x,y,z),the rang of penumbra axial length ratios of multisource static focusing modes,multi-source single-axis rotating focusing mode and single-source double-axis rotating focusing mode were 0.13-0.48,0.17-0.33 and 0.28-0.54,in the diagonal direction of the wings plane (NSD,PSD),were 0.31-0.39,0.38-0.43 and 0.54-0.72,respectively;the penumbra axial length ratio of single-source double-axis rotating focusing mode was bigger than in multi-source static focusing modes and multi-source single-axis rotating focusing mode.On the no-wings plane,the area ratios of 80％ dose curve enveloped and 50％ dose curve enveloped(A80％/A50％)were 0.40,0.47 and 0.19,on the wings plane,were 0.61,0.53 and 0.35,respectively.The field uniformity of multi-source static focusing modes and multi-source single-axis rotating focusing mode were superior to single-source doubleaxis rotating focusing mode.Conclusions Considering dose distribution of the single-shot fields,the multi-source static focusing modes devices and the multi-source single-axis rotating focusing mode devices should be preferred,when important tissues and organs are adjacent to the target areas.Compared with single-source double-axis rotating focusing mode,both multi-source static focusing modes and multi-source single-axis rotating focusing mode could make more target areas to be surrounded by high dose region.

Objective To investigate the clinical application and manifestation of dynamic contrastenhanced MRI (DCE-MRI) in differentiating true progession from pseudoprogression in patients with gliobastomas.Methods Twenty five glioma patients were treated with postoperative concurrent chemoradiotherapy and enrolled in this study.All patients were underwent DCE-MRI using a 1.5T scanner.Fifteen patients were confimmed by secondary pathology or clinical and imaging follow-up of patients with gliomas true progession (TP),10 patients were pseudoprogress (PP).Nonparametric Mann-Whitney test was used to compare perfusion parameters between two groups (TP and PP),were used for receiver operating characteristic (ROC) curve analysis to clear if these parameters can be the indicators to differentiate true progession from pseudoprogression.Results Ktrans (volume transfer constant),Ve (fractional volume of extravascular extracellular) values between TP and PP glioma groups were statistically significant,K and Ve values were significantly higher in the TP group than in the PP group (P ＜ 0.05).The areas under the ROC curve are 0.990 and 0.847,respectively.Kep (efflux rate constant) value,Vp (fractional volume of plasma) value in the identification of glioma TP group and PP group was not statistically significant (P ＞ 0.05).Conclusions DCE-MRI can be used to identify glioma TP and PP,Ktrans value and Ve value have clinical significance.

Objective To observe the effect of inducible costimulator(ICOS) costimulation pathway blockade in rat limb allografts acute rejection by RNA interference. Methods Twenty-seven cases of modified hind llmb allotransplantation were performed from Wistar to SD rats. The rats were divided into 3 gronps(each n=9): the rejection group not given a special disposal; the control group, consisting of SD rats that received injection of pSilencer 4.1 and Sofast complex by vein post transplantation; and the interference group that received injection of pSilencer 4.1-ICOSshRNA and Sofast complex. On the eighth day posttransplantation, 3 rats were killed to study the pathological changes in each group. The expressions of ICOS gene in vivo were detected by flow cytometry and RT-PCR. The mixed lymphocyte reaction (MLR) was performed and eytokines in blood were measured by ELISA. The rest rats were used to record limb survival time. Results The mean survival time in rats of the rejection and the control groups were(11.34±1.21) and (11.14±1.32) days respectively. In the interference group, the mean survival time of limb allografts was (16.85±1.73) days(P<0.05). The rats in the rejection and the control groups experienced moderate to serious acute rejections with skin epidermal necrosis, a large quantity of lymphocyte infdtration, muscle cell necrosis and interstitial edema, while the pathological changes in rats of the interference group were mild. The splenocyte ICOS mRNA expression level in the interference group(18.75%) was significantly lower than that of the rejection group(100%) and the control group(98.51%). ICOS cell surface expression level as judged by the fluorescence intensity was 45.59±12.87 in the interference group, 103.72±21.76 in the rejection group, and 93.47±29.55 in the control group(F=6.89, P<0.05). In stimulation assays, a one-way mixed lymphocyte reaction stimulation index(SI), with spleen cells from Wistar and Lewis rats, respectively, the rejection group (5.26±0.42,5.18±0.29) and the control group (5.37±0.27,4.93±0.44) had significantly greater reactions than the interference group(2.37±0.35, 4.87±0.36), respectivily(F=7.29, P<0.05; F=6.19, P0.05). In the IFN-γ and IL-4 expression assays, reactions of the interference group (230.17±38.47,160.32±59.13) were lower than those of the rejection group(490.73±51.48,230.67±45.21) and the control group(480.15±43.96, 240.53± 47.36), (F=7.23,6.75, all P<0.01). Conclusions In vivo transfection of pSilencer 4.1-ICOS shRNA interference plasmid can effectively block T-cell co-stimulation pathway, suppress acute rejection, and prolong limb allografts survival.

OBJECTIVETo verify the efficacy of Jianpi-xingniao needling therapy on prevention and treatment of motion sickness.

METHODSSixty volunteers of motion sickness were randomized into an acupuncture group and a delayed acupuncture group, 30 cases in each one. In the acupuncture group, acupuncture was given at Baihui (GV 20), Sishencong (EX-HN 1), Neiguan (PC 6), Zusanli (ST 36) and Hegu (LI 4). The needles were retained for 20 min. The treatment was given twice a week and 10 treatments were required. In the delayed acupuncture group, acupuncture was postponed, meaning no acupuncture during observation stage. Graybel scale was adopted to observe the score of symptoms and physical signs of the subjects of motion sickness before and after intervention. The efficacy was compared between the two groups.

RESULTSTwenty-five cases in the acupuncture group and 22 cases in the delayed acupuncture group were included in the statistical analysis. The score of symptoms and physical signs of motion sickness was reduced significantly after treatment in the acupuncture group as compared with that before treatment (10.12 +/- 3.37 vs 0.92 +/- 0.40, P < 0.05). The score in the acupuncture group was lower apparently than that in the delayed acupuncture group (0.92 +/- 0.40 vs 9.86 +/- 2.53, P < 0.05). The difference was not significant before and after treatment in the self-comparison of the delayed acupuncture group (P > 0.05). The total effective rate was 96.0% (24/25) in the acupuncture group, which was significantly better than 0.0% (0/22) in the delayed acupuncture group (P < 0.01).

CONCLUSIONJianpixingniao needling therapy relieves the symptoms of motion sickness in the patients and achieves a better clinical efficacy.

Acupuncture Points ; Acupuncture Therapy ; Adult ; Female ; Humans ; Male ; Motion Sickness ; therapy ; Treatment Outcome ; Young Adult

OBJECTIVETo prepare and purify recombinant human NAMPT and NAMPT (H247A) proteins and to detect their enzymatic activity.

METHODSUsing pcDNA3.1-hnampt as template, full-length hnampt was sub-cloned into pET-11a(+) plasmid. The hnampt (H247A) mutant was obtained by site-directed mutagenesis. The plasmids were introduced in Escherichia coli BL21star for protein expression. The recombined NAMPT and NAMPT (H247A) proteins were purified by flowing through nickel column and size-exclusion column. The target proteins were confirmed by SDS-PAGE and mass spectrometry detection. The enzymatic activities of recombinant proteins were assessed by solution NMR.

RESULTThe DNA sequences showed that hnampt (wild type) and hnampt (H247A) (mutation) were cloned into pET-11a(+). The recombinant proteins were expressed in Escherichia coli BL21star in soluble form. The purified protein was confirmed to be NAMPT with a molecular weight of 56 KD. The enzyme activity of NAMPT (H247A) was dramatically decreased compared to wild-type NAMPT.

CONCLUSIONThe recombinant hNAMPT and hNAMPT (H247A) proteins have been successful prepared and purified. The H247A mutation dramatically decreases the enzymatic activity of NAMPT.

Base Sequence ; Cytokines ; genetics ; isolation & purification ; metabolism ; Escherichia coli ; genetics ; Genetic Vectors ; Humans ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutation ; Nicotinamide Phosphoribosyltransferase ; genetics ; isolation & purification ; metabolism ; Plasmids ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Transformation, Bacterial

Objective with the advantages of rapidity in detection protein, We selected the gender-specific amino acid sequence based on human SMCY and SMCX, cloned and expressed SMCY gender-specific fusion antigens. The rabbits were immunized with purified antigens to obtain the polyclonal antibodies. A new method was established for rapidly sex identification of forensic evidence samples by detecting SMCY antigens with the corresponding polyclonal antibodies. Methods We found three differential fragments by analyzing the sequence of human SMCY and SMCX. Then we cloned this three fragments and ligated as a new recombinant.This SMCY gender-specific fusion antigen gene was sub-cloned into pET-28a and expressed in Escherichia coli. The fusion antigen was purified by Ni-NTA column. The rabbits were immunized with purified antigen to produce the specific polyclonal antibodies.The reactivity of the polyclonal antibody was evaluated by ELISA and Western blotting. We developed a colloidal gold test strip for detecting the gender of human samples. Results We successfully selected gender-specific amino acid sequence, the SMCY gender-specific fusion antigen was expressed by prokaryotic expression and the polyclonal antibody was prepared by immunizing rabbit. The results of colloidal gold strip tests showed that there is a significant difference between male and female serums. Conclusion The results showed that the SMCY gender-specific fusion antigen could be recognized by the polyclonal antibody.The colloidal gold strip tests made by SMCY gender-specific fusion antigens and the corresponding polyclonal antibodies could be used for rapidly determining the gender of forensic evidence samples.

OBJECTIVETo construct full-length hepatitis B core particles presenting preS1 aa 21-47 epitope and truncated core particles presenting preS1 aa 37-45 epitope on their surface and compare their antigenicity.

METHODSPreS1 aa21-47 epitope and aa 37-45 epitope were inserted respectively into full-length hepatitis B core (aa 1-183) and truncated HBcAg (aa 1-144), between the 78th (Asp) and 79th (Pro). The genes synthesized after the codon optimization were ligated to the pET43. 1a vector with the same cohesive terminal (NdeI and XhoI) and expressed in the E. coli expression system. The morphology of the proteins of interest were observed by electron microscope and characterized by ELISA and Western Blotting.

RESULTSThe morphology of the virus-like particles were confirmed by electron microscope. H2 were solid particles with a diameter of (31.61 +/- 1.27) nm, while H3 were hollow particles with a diameter of (28.46 +/- 1.16) nm. Statistical analysis showed that H2 is larger than H3 in the diameter (P < 0.01). The antigenicity of the inserted epitopes and carrier protein were identified by ELISA and Western Blotting.

CONCLUSIONChimeric hepatitis B core particles presenting the preS1 neutralizing epitopes on their surface have been expressed, purified and identified, which lays the foundation for its application in vaccine research.

Epitopes ; chemistry ; genetics ; immunology ; Hepatitis B ; immunology ; virology ; Hepatitis B Core Antigens ; chemistry ; genetics ; immunology ; Hepatitis B Surface Antigens ; chemistry ; genetics ; immunology ; Hepatitis B virus ; chemistry ; genetics ; immunology ; Humans ; Neutralization Tests ; Protein Precursors ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology

OBJECTIVETo further verify the efficacy and safety of locally manufactured lamivudine on patients with chronic hepatitis B (CHB).

METHODS2200 patients with CHB were recruited and received lamivudine orally 100 mg once daily for 12 months. The efficacy assessments included virologic response rate (defined by the absence of serum HBV DNA, HBeAg loss and HBeAg/HBeAb seroconversion), percentage of patients with normalization of alanine aminotransferase (ALT). Meanwhile improvement of quality of life (QOL) measured by mos SF-36 QOL questionnaire and liver histology evaluation were conducted in some patients. The safety assessments included adverse events, serious adverse events and laboratory abnormalities. All 2200 patients received at least one dose of medication and were all included in the safety population.

RESULTSNinety seven percent of patients (2137/2200) recruited were HBV DNA positive by dot blot (sensitivity GRT or equal to 1.0 pg/ml) at baseline. At the end of 12 months treatment, HBV DNA was undetectable in 80% patients (1538/1920) with HBV DNA positive before treatment. Among the 79%(1744/2200) of the patients recruited had positive HBV DNA accompanied abnormal ALT levels at baseline, 72% patients became ALT normal. And among the 84% (1843/2200) of the patients recruited were HBV DNA and HBeAg positive, anti-HBe negative, 16% (269/1650) patients achieved HBeAg/HBeAb seroconversion after 12 months of lamivudine treatment. The HBeAg/HBeAb seroconversion rate was positive correlation to the ALT level before treatment. A total of 304 patients completed the health-related QOL questionnaire. After 12 months treatment, lamivudine improved both their physical and mental health, especially for their mental health. 133 evaluable, paired liver biopsies were obtained for histological assessment, among whom 115 patients had abnormal ALT levels at baseline. Compared with pre-treatment most of their liver injury got alleviated (51.9%) or no further deterioration (36%), only 12% worsening. During the 12 months treatment, 9% patients withdrew from the study and 17% patients showed at least one adverse event, mild or moderate. There were no obvious difference between this study and the previously reported lamivudine Phase II or III study with regard to the kinds, incidence and severity of adverse events.

CONCLUSIONThe efficacy and safety profile of the locally manufactured lamivudine 100 mg tablets are similar with those of the previously reported available lamivudine tablets imported in treating Chinese chronic hepatitis B patients.

Adolescent ; Adult ; Aged ; Antiviral Agents ; therapeutic use ; Child ; DNA, Viral ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B, Chronic ; drug therapy ; psychology ; virology ; Humans ; Lamivudine ; adverse effects ; therapeutic use ; Liver ; pathology ; Middle Aged ; Quality of Life