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ObjectiveTo establish an High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry （HPLC-ICP-MS） method for determination of six arsenic species in human urine，including arseniccholine （AsC）， arsenobetaine （AsB）， arsenite （As3+）， dimethylarsinic acid （DMA5+）， monomethylarsonic acid （MMA5+）， and arsenate （As5+）. MethodsThe pH value of mobile phase and the content of anhydrous ethanol were optimized. Ammonium carbonate （50 mmol·L-1， containing 2% anhydrous ethanol， pH-8.5） mobile phase was selected. Cl- interference was eliminated by He mode. The arsenic species in 10-fold diluted human urine samples were separated by an Hamilton PRP X-100 anionic column. A method for the determination of six arsenic species was established. ResultsSix arsenic species could be separated in 13 minutes. The linear correlation coefficients were above 0.999. The limits of detection were 0.10‒0.20 μg·L-1， and the limits of quantification were 0.30‒0.50 μg·L-1. Precision experiments showed that RSD ranged from 5.96% to 9.07% when adding concentration 0.20 μg·L-1； from 2.48% to 6.38% when adding concentration 2.00 μg·L-1； and from 1.41% to 2.57% when adding concentration 5.00 μg·L-1. Accuracy test showed that the recoveries were 80%‒125%. ConclusionThe established HPLC-ICP-MS method for determination of six arsenic species in human urine is rapid， accurate and sensitive. It can be applied to the determination of arsenic species in human urine.
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Adjuvants enhance the efficacy of the vaccine by regulating or enhancing the immune response to antigens,helping to reduce the amount of antigen and improve the vaccine safety.With the deepening of adjuvant researches,it has been found that the combination of two or more adjuvants can produce synergistic effects by activating multiple immune mechanisms.Highly effective vaccines are urgently needed for the group with pathogens that are difficult to remove and with low immune system function.This requirement can be met by the combination of immunologic adjuvants.In this paper,the progress of some new adjuvants and the applications of combined adjuvants were reviewed including liposomes,immunostimulating complexes,Montanides,polycationic peptide IC31,nanoemulsion and AS adjuvant systems.
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Major causes of cholestasis include abnormal bile metabolism, obstructed bile flow and regurgitation, and bile duct injury and obstruction. Abnormal bile metabolism is mainly caused by gene abnormality, while obstructed bile flow is often caused by bile duct injury or occlusion due to cholangitis. Abnormal bile metabolism is the most common cause of progressive familial intrahepatic cholestasis (PFIC), while autoimmune cholangitis is the major cause of bile duct injury. Both congenital/autoimmune bile duct injury and secondary/acquired ductular reaction had relatively specific histopathological changes, and a confirmed diagnosis needs a comprehensive analysis based on clinical, pathological, imaging, immunological, and genetic examinations. This article elaborates on the research advances in the pathomorphology of autoimmune bile duct injury and PFIC and introduces the similar lesions, in order to provide a reference for the clinical diagnosis of bile duct injury and cholestatic liver diseases.
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In recent years,cancer immunotherapy has developed rapidly due to its significant advantages compared with the traditional cancer treatment methods.Tumor immunotherapy aims at mobilizing or stimulating the body's own immune function,thereby inhibiting and killing cancer cells.With the development of nanotechnology,biological nano-carrier materials provide a new insight into the vaccine development.Nano-vaccines are therapeutic or prophylactic vaccines based on nanotechnology including exogenous antigens for inducing immune responses,vectors delivering antigens,and adjuvants for enhancing immunogenicity and accelerating and prolonging the availability of cancer vaccines.Nano-delivery vectors have good biocompatibility as well as unique physical and chemical properties.They can effectively deliver the antigens,and further activated the immune response of antigenspecific cellulars based on the activation of the body's humoral immunity by regulating the presentation pathways in the antigen-presenting cells.In this paper,the applications of nano-delivery systems in cancer vaccine research were summarized.
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Immunological checkpoints are a mechanism evolved by human beings to control the intensity and duration of immunoreaction and minimize the excessive inflammatory responses and autoimmune diseases caused by overactive immune responses.Compared to radiotherapy,chemotherapy and other traditional treatments,immunotherapy has fewer side effects on normal cells,and has become an emerging technology in tumor treatments.As the focus on tumor treatment research,immunological checkpoint therapy can damage the tumor cells by breaking the immune tolerance and activating the body's own immune system,which make it a promising treatment method.In this paper,the mechanisms of immune activation,immune regulation and immune evasion were described.The action mechanism and clinical application of anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death receptor-1 (PD-1) were summarized.The application prospect of immune checkpoint inhibitors was discussed.
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In recent years,cancer immunotherapy has developed rapidly due to its significant advantages compared with the traditional cancer treatment methods.Tumor immunotherapy aims at mobilizing or stimulating the body's own immune function,thereby inhibiting and killing cancer cells.With the development of nanotechnology,biological nano-carrier materials provide a new insight into the vaccine development.Nano-vaccines are therapeutic or prophylactic vaccines based on nanotechnology including exogenous antigens for inducing immune responses,vectors delivering antigens,and adjuvants for enhancing immunogenicity and accelerating and prolonging the availability of cancer vaccines.Nano-delivery vectors have good biocompatibility as well as unique physical and chemical properties.They can effectively deliver the antigens,and further activated the immune response of antigenspecific cellulars based on the activation of the body's humoral immunity by regulating the presentation pathways in the antigen-presenting cells.In this paper,the applications of nano-delivery systems in cancer vaccine research were summarized.
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Immunological checkpoints are a mechanism evolved by human beings to control the intensity and duration of immunoreaction and minimize the excessive inflammatory responses and autoimmune diseases caused by overactive immune responses.Compared to radiotherapy,chemotherapy and other traditional treatments,immunotherapy has fewer side effects on normal cells,and has become an emerging technology in tumor treatments.As the focus on tumor treatment research,immunological checkpoint therapy can damage the tumor cells by breaking the immune tolerance and activating the body's own immune system,which make it a promising treatment method.In this paper,the mechanisms of immune activation,immune regulation and immune evasion were described.The action mechanism and clinical application of anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death receptor-1 (PD-1) were summarized.The application prospect of immune checkpoint inhibitors was discussed.
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Objective To investigate the effect of early continuous veno-venous hemofiltration (CVVH) on intra-abdominal pressure (IAP) and serum interleukin-6 (IL-6) in severe acute pancreatitis (SAP) patients with abdominal compartment syndrome (ACS).Methods 41 SAP patients with ACS were selected as treatment group and treated with CVVH as well as conventional methods in ICU.The other 12 patients with the same disease were selected as the control group and were only treated with conventional methods because of economic reasons.IAP and blood level of IL-6 in the two groups were measured daily in order to investigate their changes and the correlation between the two parameters.Results The serum IL-6 level and IAP in the two groups were higher on admission day.IAP and serum IL-6 level in the treatment group were significantly decreased on the first day after treatment,and thereafter decreased rapidly.In the control group,IAP and serum IL-6 level were significantly decreased on the 3rd day after treatment.IAP and serum IL-6 level from the 1st day to the 6th day after treatment in the treatment group were significantly lower than those of the control group at the same time point (P＜0.05).There was a significant positive correlation between blood IL-6 level and IAP in SAP patients with ACS(r=0.48,P＜0.01).IL-6 difference before and after treatment was also positively correlated with the difference of IAP(R=0.39,P＜0.05).Conclusions VVH significantly decreased the IAP and the blood level of IL-6 in ACS patients of SAP.The blood level of IL-6 is significantly correlated with IAP,suggesting that IL-6 may play an important role in the pathogenesis of ACS.Therefore early CVVH may clear the cytokines such as IL-6 and lower IAP,thus to prevent multiple organ dysfunction syndrome (MODS),which should be applied in the early stage of ACS.
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Objective To explore the effects of surface functionalized multi-walled carbon nanotubes (FMWCNTs) on the cytotoxicity of human peripheral blood mononuclear cell (PBMC).Methods Five different types of MWCNTs (hydroxylated,carboxylated,aminated,nickel-plated and pristine MWCNTs (P-MWCNTs)) with the same diameter and length were evaluated the dispersion and characterizations in physiological salt solution by transmission electron microscopy.PBMC were isolated by density gradient centrifugation from human peripheral blood,and 5 types of MWCNTs were ultrasonically dispersed in serum-containing medium respectively.After incubation with PBMC for 12,24,48 or 72 h,cytotoxicity was detected by CCK-8 kits.Results All the MWCNTs had well dispersion,especially the F-MWCNTs.Cytotoxicity results showed that all types of MWCNTs could induced PBMC death,and presented dose-dependence manner and a certain degree of time-dependence manner.Compared with the P-MWCNTs,F-MWCNTs changed cytotoxicity statistically,with the hydroxylated,carboxylated,aminated MWCNTs weakened,aminated MWCNTs significant (P＜0.05),nevertheless the nickel-plated MWCNTs increased.Compared with the P-MWCNTs (25 μg/ml),cell viability of PBMC after 24 and 48 h incubation with the same dose of nickelplated MWCNTs both decreased,and the differences was statistically significant (P＜0.01,P＜0.05).Conclusions The functional group modification affects not only the MWCNTs dispersion in medium,but also the cytotoxicity of the MWCNTs on PBMC.
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Objective To prepare stable aqueous dispersions of chitosan/multi-walled carbon nanotubes (CS/MWCNTs) composites,and observe the effects of CS/MWCNTs on the growth of human umbilical vein endothelial cells (HUVEC).Methods CS/MWCNTs composites were prepared by electrostatic interactions between negatively charged MWCNTs and positively charged low-molecular-weight CS.The prepared CS/MWCNTs were characterized by transmission electron microscopy and Zetasizer nano-analyser.The cellular uptake of the fluorescently labeled CS/MWCNTs was observed by laser confocal microscopy after incubating with HUVEC for 24 h at different concentrations.In vitro cytotoxicity and cellular reactive oxygen were also detected.Results When the mass ratio of low-molecular-weight CS to MWCNTs was equal or greater than 10∶1,the CS/MWCNTs can be stabilized in solution.Cellular uptake experiments showed that the CS/MWCNTs could enter into the cells and locate mainly in the cytoplasm.Cytotoxicity study showed that the CS/MWCNTs composites was less toxic than MWCNTs alone at high concentration (10 and 20 μg/ml).However,there was no significant differencein the level of cellular reactive oxygen between the two groups (P＜0.05).Conclusions CS/MWCNTs composites showed low cytotoxicity and high stability,which would be a promising carrier for drug delivery.
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Objective To analyze the effect of dexmedetomidine hydrochloride injection on patients with craniocerebral disease who has no artificial airway in the process of bronchoalveolar lavage treatment.Methods Forty-six patients (age 17-28,average age 56.6±9.2,26 men and 20 women) with craniocerebral disease who has no artificial airway were selected,and were treated by bronchoalveolar lavage for lung infection.The patients were randomly divided into two groups,control and test group.The control group (n=23) received midazolam for sedative and the test group (n=23) received dexmedetomidine hydrochloride for sedative while they were in the process of bronchoalveolar lavage treatment.Heart rate,mean arterial pressure and blood oxygen saturation of fingers collected from patients before and during the process of bronchoalveolar lavage were compared.Results In the process of bronchoalveolar lavage treatment,the minimum blood oxygen saturation of finger artery from the control group was lower than that from the test group,the fastest heart rate from the control group was greater than that from the test group,and the lowest mean arterial pressure from the control group was lower than that from the test group (P＜0.05).In two groups,heart rate in the process of bronchoalveolar lavage treatment was faster than that from before the treatment,while both mean arterial pressure and blood oxygen saturation of finger artery were decreased (P＜0.05).Conclusions Continuous intravenous pumping of dexmedetomidine hydrochloride on patients with craniocerebral disease who has no artificial airway during the process of bronchoalveolar lavage treatment is effective and safe,and it has less inhibitory effect on respiratory function and blood pressure.
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Objective To investigate the change of serum osteoprotegerin level and relationships between serum osteoprotegerin level and coronary heart disease (CHD) in patients with type2 diabetes mellitus (DM) complicated with CHD.Methods One hundred and eight patients with type 2 DM complicated with CHD were selected as DM with CHD group,including 68 cases with acute coronary syndrome and 40 cases with stable angina pectoris.In addition,60 type 2 DM patients without CHD (DM without CHD group) and 40 healthy people (control group) were selected.Serum osteoprotegerin level was measured by enzyme linked immunosorbent assay.Results The serum osteoprotegerin level in DM with CHD group was significantly higher than that in DM without CHD group and control group [(4.12 ± 0.71)ng/L vs.(2.69 ± 0.52) and (2.14 ± 0.37) ng/L],and there were statistical differences (P＜ 0.05 or ＜ 0.01).In DM with CHD group,the serum osteoprotegerin level in acute coronary syndrome was significantly higher than that in stable angina pectoris [(4.56 ±0.92) ng/L vs.(3.61 ±0.76) ng/L],and there was statistical difference (P ＜ 0.05).The serum osteoprotegerin level in patients with Gensini score ＞ 40 scores (41 cases)was significantly higher than that in patients with Gensini score 20-40 scores (53 cases) and patients with Gensini score ＜ 20 scores (14 cases) [(4.92 ± 0.89) ng/L vs.(4.08 ± 0.75) and (3.39 ± 0.85) ng/L],and there were statistical differences (P ＜ 0.01 or ＜ 0.05).The serum osteoprotegerin level in patients with Gensini score 20-40 scores was significantly higher than that in patients with Gensini score ＜ 20 scores,and there was statistical difference (P ＜0.05).The serum osteoprotegerin level in single-vessel lesion (16 cases),double-vessel lesion (57 cases) and three-vessel lesion (35 cases) [(3.52 ± 0.82),(4.54 ± 0.68),(4.75 ±0.93) ng/L] were significantly higher than those in control group,and there were statistical differences (P ＜ 0.05); and the serum osteoprotegerin level in double-vessel lesion and three-vessel lesion were significantly higher than those in single-vessel lesion,and there were statistical differences (P ＜ 0.05) ;there was no statistical difference between three-vessel lesion and double-vessel lesion(P＞ 0.05).The results of Logistic regression analysis showed that glycosylated hemoglobin,total cholesterol,high sensitivity C reactive protein and osteoprotegerin were independent risk factors of type 2 DM complicated with CHD.Conclusions Serum osteoprotegerin level in patients with type 2 DM complicated with CHD is significantly increased.There is a significant association between serum osteoprotegerin level and the presence and severity of CHD,and the serum osteoprotegerin level is independent risk factors of type 2 DM complicated with CHD.
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Objective To detect the size distribution and Zeta potential of LHRH-MPG△NLS/CDK-siRNA nanoparticles,to observe the effect of different solvents on the nanoparticle size,and to investigate the inhibitory effect of nanoparticles on HepG2 cell growth.Methods LHRH-MPG △NLS and CDK2-siRNA were mixed by continuous stirring to form nanoparticles at different N/P ratios (10/1,20/1 and 40/1).The size distribution and Zeta potential of LHRH-MPG△NLS/CDK2-siRNA nanoparticles were detected by dynamic light scattering,and the stability of the nanoparticles in normal saline,10％ glucose and pure water was discussed.Finally,the inhibitory effect of the nanoparticles on HepG2 cells was determined by CCK8 kit.Results The mean size of the nanoparticles was within 200 nm,and the Zeta potentials were (70±5) mV (N/P=10/1),(120±5) mV (N/P=20/1) and (130±5) mV (N/P=40/1),respectively.The size of the nanoparticles in normal saline was significantly increased,which demonstrated that strong electrolytes had a great impact on the nanoparticles size.When nanoparticle concentration was 200 nmol/L,LHRH-MPG△NLS/CDK2-siRNA nanoparticles (N/P=10/1) showed significantly inhibitory effect on HepG2 cell growth.Conclusions The mean size of the LHRH-MPG△NLS/CDK2-siRNA nanoparticles was within 200 nm,which was ideal for cellular uptake.The Zeta potential of nanoparticles revealed that nanoparticles could be stable in aqueous solution,while strong electrolytes would affect nanoparticle size.When nanoparticle concentration was 200 nmol/L,LHRH-MPG△NLS/CDK2-siRNA nanoparticles (N/P=10/1) showed significantly inhibitory effect on HepG2 cell growth.
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Objective To investigate the cell compatibility of polystyrene(PS) plate chemically modified with RGD peptides.Methods PS surfaces were carboxylated by permanganate oxidation in diluted sulfuric acid,and carboxyls were activated with water-soluble carbodiimide to graft with gelatin,collagen and RGD peptides.IR,X-ray photo-electronic spectroscopy (XPS) and dynamic contact angle were used to characterize the surface modification of PS surface.Results XPS results confirmed the existence of nitrogen element from protein molecules and the covalently binding of proteins to PS surfaces.Dynamic contact angle measurement indicated hydrophilicity of PS surfaces was improved obviously after grafting modification.The cell culture results showed that the cell adhesion and proliferation was better on modified surfaces than the initial.Conclusion The cell compatibility of PS surface was great improved after modification with RGD peptides,which would provide a potential strategy to improve the culture of purified endothelial progenitor cells isolated by immunomagnetic beads.
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ObjectiveTo investigate the cytotoxicity and gene transfection mediated by NMPCS/DNA nanoparticles.MethodsN-methylene phosphonic chitosan (NMPCS) was synthesized using one-step reaction under homogeneous conditions.The NMPCS/DNA nanoparticles were prepared using complex coacervation method.The cytotoxicity of NMPCS alone and its complexes with plasmid DNA were determined by MTT assay on HeLa cells.The gene transfection mediated by NMPCS/DNA nanoparticles were investigated using pGL3control vector as reporter gene.ResultsThe MTT results suggested that the NMPCS and NMPCS/DNA complexes showed significantly lower cytotoxicity than PEI and PEI/DNA complexes,respectively.The gene transfection mediated by NMPCS/DNA nanoparticles were greatly improved compared with unmodified chitosan.ConclusionNMPCS would demonstrate great potential as a novel,safe,efficient non-viral vector for gene delivery.
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ObjectiveTo explore the endocytic pathway of TAT-LHRH modified chitosan/DNA nanoparticle (TLCDN) that exhibits high transfection efficiency and targeting to HepG2.MethodsPlasmid DNA was labeled with fluorescein,and the resulting fluorescent DNA was complexed with chitosan or TAT-LHRH modified chitosan to form chitosan/DNA nanoparticle (CDN) and TLCDN by the complex coacervation method.Internalization of TLCDN or CDN by HepG2 cells were measured in the presence of three kinds of inhibitors of endocytic pathway,Chlorpromazine,Filipin or Dynasore,using High-Content Analyzer to collect and analyze the data.ResultsChlorpromazine led to more decreased uptake of CDN than that of TLCDN,although not statistically significant.Filipin demonstrated significant inhibitory effect on the uptake of TLCDN while promoted the uptake of CDN.Dynasore resulted in a similar decrease in the uptake of both nanoprticles.ConclusionIt was demonstrated that CDN was taken up by HepG2 cells mainly through the clathrin-dependent endocytic pathway and TLCDN was more likely to be internalized by HepG2 cells through the caveolin-mediated endocytic pathway although the clathrin-dependent endocytic pathway was also involved.
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ObjectiveConjugation of fluorescent dye onto plasmid DNA was investigated in order to monitor delivery process of plasmid DNA.MethodsPlasmid was activated with bromine,stored for different timeintervals at 4 ℃ or room temperature,and subsequently coupled with 1,10-diaminodecane to prepare aminemodified plasmid DNA.Amine-modified plasmid was then reacted with isothiocyanate (FITC) for fluorescent labeling,and the labeling ratio was calculated after purification.The effect of storage conditions (time/temperature) of bromine-actived plasmid (BP) on fluorescent labeling efficacy was estimated,and the cell transfection efficiency of fluorescent plasmid-lipofectamine complex was observed.The fluorescent plasmid delivered by lipofectamine 2000 in A10 cells was observed by laser scanning confocal microscope (LSCM) and flow cytometry.ResultsThe experimental data showed that prolonged storage time of bromine-activated DNA had a negative effect on the labeling ratio,and lower storage temperature had a positive effect on the labeling ratio.It also demonstrated that FITC modification had no effect on the transfection efficiency of plasmid-lipofectamine complex as compared with that of unlabeled plasmid-lipofectamine complex,and FITC modified plasmid had enough fluorescent intensity to monitor cell uptake with flow cytometer and sub-cellular distribution with LSCM.ConclusionA facile method for conjugating fluorescent dye onto plasmid was established in the study,and could be utilized to trace the plasmid delivery for investigating the transfection mechanism.
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ObjectiveTo isolate and identify endothelial progenitor cells (EPCs) from human umbilical cord,and to study the cell proliferation and gene transfection of green fluorescent protein plasmid in vitro.MethodsEPCs were isolated from human umbilical cord in enzyme digestion method.The biological characteristics of EPCs were identified by flow cytometry and laser confocal microscope.The enhanced green fluorescent protein (EGFP) gene transfection mediated by EPCs was investigated using Lipofectamine 2000 as transfection reagent.ResultsEndothelial progenitor cells isolated from umbilical cord formed typical endothelial cell colony 9 days later.These cellsdisplayed an improved positive expression of CD133 and kinase insert domain receptor (KDR).The endotheliallineage characteristics of expanded cells were confirmed by fluorescein isothiocyanate (FITC)-UEA-1 binding and DiI-ac-LDL uptake assay with the aid of laser confocal microscope.The transfection results demonstrated high expression of EGFP taking EPCs as host cell.ConclusionEndothelial progenitor cells isolated from umbilical cord can be propagated and induced to differentiate into endothelial cells in the appropriate culture conditions.EPCs demonstrated to be an ideal carrier for gene and cell therapy.
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Objective To generate recombinant Pichia pastoris for high-copy expression of human tissue factor pathway inhibitor (TFPI). Methods The cDNA encoding human TFPI was inserted into the expression vector pPIC9K and the constructed expression vector rhTFPI-pPIC9K was confirmed by restriction endonuclease analysis and DNA sequencing. The recombinant plasmids were subsequently transformed into Pichia pastoris GS115 cells, and the transformants were confirmed by PCR amplification of the genomic DNA.The recombinant Pichia pastoris with high copies of TFPI cDNA was screened by G418 selection. Western blot and TFPI ELISA Kit were employed to analyzing. The temperature, time and concentration of methanol for the induction of recombinant protein were optimized. Results PCR analysis and DNA sequencing confirmed the successful construction of the expression vector rhTFPI-pPIC9K. Real time quantitative PCR and Western blot analysis demonstrated the positive correlation between TFPI expression level and the copy number of TFPI cDNA in Pichia pastoris cells. Optimization of the induction condition significantly elevated the expression level and activity of TFPI (9.95±0.78 mg/L and 3.91±1.37 U/mL). Conclusion The Pichia pastoris strain with high copy of TFPI expression was successfully constructed, which lays a solid foundation for the further investigation on the function of TFPI and its application in the prevention and therapy of diseases.
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Objective To investigate the impact of chitosan and alkylated chitosan DNA nanoparticles on the function of human naive CD4+T cells.Methods The secretion of cytokines (IL-4 and TNF-γ) was observed after the co-incubation of human naive CD4+T cells with nanoparticles 12 h,24 h and 48 h,respectively.ResultsNone of the nanoparticles induced the production of cytokines ( IL-4 and TNF-γ ).Conclusion Chitosan and alkylated chitosan DNA nanoparticles will not induce the differentiation of human naive CD4+ T cells into T1 or T2 and may be considered as a safe gene carrier.