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Galectin-3 (Gal-3) belongs to the galectin family and is specific in binding β-galactoside. Through its C-terminal domain, Gal-3 binds to the galactoside group of the glycosylated insulin receptor (IR) and inhibits IR signaling pathway, which leads to the insulin resistance. Thus, Gal-3 is a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes. Here we report a simple Gal-3 screening model based on the property that Gal-3 binds to the galactoside. We expressed and purified human Gal-3 in Escherichia coli (E.coli), and labeled it with fluorescein isothiocyanate (FITC) in vitro. After incubating FITC labeled Gal-3 (Gal-3-FITC) with PANC-1 cells, which express glycosylated membrane protein, PANC-1 cells started to show green fluorescent signal due to the Gal-3-FITC binding to the glycosylated membrane protein. Gal-3 inhibitor disrupts the binding of Gal-3-FITC and PANC1 cells, subsequently leads to the decrease of the fluorescent signal in PANC-1 cells. We can evaluate the inhibitory efficiency of Gal-3 inhibitors through measurement of the fluorescent signal. Further studies show this model is simple, stable, and repeatable with a Z' factor between 0.7 and 0.85. In sum, we have successfully established an in vitro high-throughput screening model for Gal-3 inhibitors.
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【Objective】 To establish a new method for the determination of fibrinogen content in cryoprecipitated antihemophilic factor. 【Methods】 Fibrinogen (Fib) could bind with sheep anti-human fibrinogen (anti-Fib) specifically and further form antigen-antibody complex. When the Fib was present in the solution, the fluorescence of fluorescein isothiocyanate (FITC) labeled on the anti-Fib (FITC-anti-Fib) was quenched due to the formation of immune complex. The fluorescence quenching degree of FITC-anti-Fib was positively correlated with Fib concentration (cFib) in a certain concentration range. 【Results】 The linear relationship between fluorescence quenching degree [(I0-I)/I0] of FITC-anti-Fib and ln(cFib) was (I0-I)/I0=15.53ln(cFib)+ 80.79 (R2=0.99) when the cFib was in the range of (0.007 8-0.560 0) g/L. The recovery of Fib was (96.77-102.43) %. When the method was applied to determine Fib at high, medium, and low concentrations, the obtained intra-day variation coefficients were 0.31%, 0.56%, and 0.49%, respectively, and the inter-day variation coefficients were 3.81%, 3.06%, and 4.13%, respectively. There was no significant difference between the results measured by fluorescence quenching method and coagulation method (t=-0.075, P>0.05). 【Conclusion】 In this work, a new fluorescence method for the determination of Fib in cryoprecipitated antihemophilic factor was successfully established based on the specific combination of fib and FITC-anti-Fib. The method is simple and rapid. The obtained results were accurate and reliable by using this method to determine Fib.
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Objective: To explore the factors affecting the nasal entry of the pharmaceutical preparations into the brain based on the established model of the "nose-brain" pathway in vitro. Methods: Calu-3 cells and OECs cells were co-cultured to construct a "nasal-brain" pathway cell model group. Taking fluorescein isothiocyanate dextran (FD) and fluorescent silver nanoparticles (AgNPs) as model drugs, the effects of drug molecular weight (Mw) factors and preparation particle size factors on the drug transnasal transport into the brain were explored. Results: The apparent permeability coefficient (Papp) of transcellular monolayer transport of FD decreased with the increase of molecular weight. The uptake of fluorescein isothiocyanate dextran with different molecular weights by OECs tended to be saturated after 90 min. As the molecular weight of FD increased, the uptake of OECs decreased significantly during the same uptake time. The apparent permeability coefficient of fluorescent AgNPs with different particle sizes in the "nose-brain" multi-channel cell model group of calu-3 monolayer decreased with the increase of the particle size of the nanoparticles. When the particle size was less than 40 nm, its transport characteristics in Calu-3 were shown as medium absorption (1 × 10-6 < Papp < 10 × 10-6), and when the particle size of nanoparticles was more than 60 nm, its transport characteristics were shown as difficult to absorb (Papp < 1 × 10-6). The uptake of OECs of fluorescent AgNPs with different particle sizes tended to be saturated at 60 min, and with the increase of the particle size of fluorescent AgNPs, the uptake of OECs at the same uptake time showed a significant decline. Conclusions The Mw of the drug and the particle size of the nano-formulation have an important influence on the nasal transport of the drug into the brain. Drugs with a molecular weight of < 4 000 and nano particles with a particle size of less than 40 nm have better transport and uptake characteristics.
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OBJECTIVE: To prepare conjugate of low molecular weight chitosan(LMWC) with glycyrrhizin (GL) and investigate its cellular uptake by proximal tubular epithelial cells (HK-2). METHODS: Glycyrrhizin-LMWC (GL-LMWC) conjugate was synthesized by the reaction between the amino group of LMWC and the active ester group of GL. Drug release was studied in phosphate buffer (pH 4.0) containing papain. The uptake of the conjugate by HK-2 cells and its intracellular distribution was studied with laser confocal microscopy. RESULTS: The chemical structure of GL-LMWC conjugate was confirmed by IR and H-NMR. The GL grafting rate was determined to be 29.3%. The drug liberation from the conjugates was significantly accelerated in the presence of papain, which confirmed the capability of the conjugate to degrade in lysosomes. The conjugate could be internalized by HK-2 cells. CONCLUSION: The GL-LMWC conjugate with well-defined structure is successfully synthesized and exhibitts high cellular uptake efficiency. The conjugate has the potential to retaing GL in the kidney for a prolonged duration and to sustain its release locally for better efficacy.
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Objective To investigate the protective effect of allicin on intestinal mucosal barrier of septic rats so as to explore the possible mechanism.Methods Twenty-four male SD rats were randomly (random number) divided into sham,septic model and allicin treatment group.Septic model was established by cecal ligation and puncture (CLP) in rats.Rats in the treatment group were administered with allicin (30 mg/kg,ip)at 6 h and 12 h after modeling,while those in the model and sham groups were treated with equal amount of saline instead.Rats were sacrificed at 24 h and the serum D-lactic acid,diamine oxidase (DAO) and fluorescence isothiocyanate-dextran (FITC-Dextran,FD-40) were determined to evaluate the intestinal mucosal barrier function.The levels of tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6),malondialdehyde (MDA),and the activity of superoxide dismutase (SOD) in intestinal tissue were measured.Histopathological changes of intestinal mucosa injury were assessed by Hematoxylin-eosin staining.Results Compared with the sham group,levels of serum D-lactic acid,DAO and FD-40 increased significantly in the CLP group (D-lactic acid:599.4±101.1 vs.149.2±20.63 nmoL/mL,t=11.84,P＜0.01;DAO:302.1 ±64.5 vs.76.57±14.76 ng/mL,t=9.433,P＜0.01;FD-40:6664.0±1437.0vs.1446.0±205.0 ng/mL,t =9.704,P ＜0.01);intestinal morphology damage occurred in the CLP group;intestinal levels of TNF-α,IL-6 and MDA increased greatly (TNF-αt:186.35 ±20.43 vs.58.76 ±8.94 pg/mL,t=17.23,P＜0.01;IL-6:763.25±85.23vs.125.36±14.37 pg/mL,t=22.54,P＜0.01;MDA:29.36±3.27vs.7.24±0.85 nmol/mg prot,t=16.61,P＜0.01),while SOD activity reduced (35.75±6.53 vs.73.26 ±8.35 U/rmg prot,t =10.57,P ＜0.01) in the CLP group.Allicin treatment greatly inhibited the increase of D-lactic acid,DAO and FD-40 levels in rat plasma caused by CLP (D-lactic acid:330.1 ±81.77 vs.599.4±101.1 nmol/mL,t=7.086,P＜0.01;DAO:171.8±49.70vs.302.1±64.56ng/mL,t=5.45,P＜0.01;FD-40:3349.0±1167.0 vs.6664.0±1437.0 ng/mL,t=6.165,P＜0.01);intestinal morphology damage was improved in the allicin treatment group;allicin treatment greatly inhibited the intestinal levels of TNF-o,IL-6 and MDA and preserved the intestinal SOD activity compared with the CLP group (TNF-α:95.37 ±12.68 vs.186.35 ±20.43 pg/mL,t =12.29,P＜0.01;IL-6:354.27±46.27vs.763.25±85.23pg/mL,t=14.45,P＜0.01;MDA:16.27±3.14vs.29.36±3.27 nmol/mgprot,t=9.831,P＜0.01;SOD:55.35 ±6.23vs.35.75±6.53 U/mgprot,t=5.522,P ＜0.01).Conclusions Allicin could inhibit local inflammation and oxidative stress in the intestine and exerts protective effect on intestinal mucosal barrier of septic rats.
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Objective Quadratic regression universal rotary combination design was used to optimize the fluorescence labeling condition of lycium barbarum polysaccharide (LBP).Methods The fluorescence labeling condition of LBP presented, when the covalent coupling of LBP and tyramine was reacted with fluorescein isothiocyanate (FITC). Filter the best labeling condition via using quadratic regression universal rotary combination design experiment on the relationship of labeling efficiency among pH value of buffer solution, reaction time, temperature and the dose of tyramine.Results The regression equation was:Y=0.085 41 - 0.002 82X1 - 0.015 68X2 + 0.008 11X3 + 0.005 01X4 + 0.008 75X1X2 - 0.005 75X1X3 - 0.001 75X1X4 + 0.010 63X2X3 + 0.000 125X2X4 + 0.000 25X3X4 - 0.021 44X12 - 0.008 89X22 - 0.001 984X32 + 0.003 66 X42, and the variables fromX1 toX4 represented pH value, reaction time, temperature and dose of tyramine, respectively. The goodness of fitting of regression equation was statistically significant. The condition of labling LBP was optimized when the temperature was at 50℃, timing of labling at the fourth day and pH value was 8.5.Conclusion The condition of labling LBP was optimized with suitable temperature, pH value and extended timing.
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Objective To investigate the characters and targeted ability of FITC-CSNRDARRC molecular probe in labeling orthotopic transplantation tumor of bladder cancer in vivo.Methods From July 2013 to June 2014,the stability and characters of FITC-CSNRDARRC molecular probe were detected by spectrophotometer and molecular imaging and the optimum concentration and imaging time window were determined.30 BALB-C nude mice were randomly divided into experimental group (n =20) and control group(n =10).In control group,5 of them (group A) were ligated bilateral ureter,others(group B) were not.We established orthotropic transplanted bladder tumor (BIU-87) model by operation.And 0.2 ml probes (220 μmol/L) was then injected intravenously in all mice after 2 weeks.We obtained images and analyzed average gray value of the heart,lung,liver,spleen,bilateral kidney and orthotropic transplantation bladder tumor by using optical probe molecule fluorescence imaging system after 30 min,1 h,2 h,4 h and 12 h,respectively.Results After injected the FITC-CSNRDARRC molecular probes intravenously at 220 μmol/L,the fluorescence signal of tumor tissue strengthened gradually.The optimal imaging time window was 4 hours after injection.The illumination and temperature had little effect on the fluorescence signal.With the time passing after injection,the intensity of florescence signal progressively increasing,which reached the peak at4 h.The average gray value of tumor tissue at 1 h,2 h,3 h,4 h,5 h,6 h,8 h,12 h were 74.22,76.2,80.11,89.38,83.29,85.1,81.22,83.01,respectively.The fluorescence signal of normal tissue weakened gradually with the passage of time.Only liver and gall bladder could notice the fluorescence signal 4 hours after injection in group A.However,the relatively strong fluorescence signal could be found in liver and gall bladder in group B.Conclusions The characters of fluorescence probe are affected by its concentration.Its optimal concentration of labeling tumor is 220 μmol/L.The optical imaging time window was about 4 h after intravenous injection.The FITC-CSNRDARRC molecular probe can specifically bound to orthotopic transplanted tumor of bladder cancer in vivo.
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Objective: To synthesize butyryl galactose ester (But-Gal) and prepare butyryl galactose ester-modified coix component microemulsions (But-Gal-CMEs) and to evaluate its physicochemical properties and anticancer activity in vitro. Methods: But-Gal was synthesized by enzyme-catalyzed reaction and the structure of the product was confirmed by 1H-NMR and FT-IR. The CMEs and But-Gal-CMEs were prepared by aqueous titration method using coix seed oil, Cremophor RH40, PEG400, But-Gal, and coixan solution as oil phase, surfactant, cosurfactant, target ligand, and aqueous phase, respectively. The average particle size, polydispersity index (PDI), and Zeta potential were detected by dynamic light scattering (DLS). The cytotoxicity of CMEs and But-Gal-CMEs aganist HepG2 cells was determined by MTT assay. The cellular uptake of CMEs and But-Gal-CMEs was detected by fluorescence microscopy. Results: The structure of But-Gal was confirmed by 1H-NMR and FT-IR. The But-Gal-CMEs displayed the spherical surface with mean droplet size of (57.68 ± 6.65) nm, PDI of 0.070 ± 0.006, and Zeta potential of (-2.95 ± 0.23) mV, respectively. MTT experiments showed that the half of HepG2 cell proliferation inhibition concentration (IC50) of But-Gal-CMEs and CMEs was 62.55 and 71.23 μg/mL. The HepG2 cell uptake results suggested that the fluorescence intensity of But-Gal-CMEs group was higher than that of CMEs group. Conclusion: The But-Gal-CMEs presents small particle size, good roundness, and good stability. In addition, But-Gal could increase the uptake rate of CMEs in HepG2 cells and enhance the inhibition of HepG2 cell proliferation.
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Background The incidence of retinal vascular diseases increase annually,such as diabetic retinopathy,retinopathy of prematurity and age-related macular degeneration.The key of treatment for these diseases is how to evaluate retinal vascular change effectively and objectively.Retro-orbital injection of fluorescein isothiocyanatedextran (FITC-dextran) is a simple and effective method for observing C57BL/6J mouse retinal vessels.But,whether it is suitable for other mice and rats is seldom reported.Objective This experiment was to assess the feasibility of the observation of retinal vessels by retro-orbital injection of FITC-dextran in different genus of mouse and offer the reference for relevant study.Methods Twelve animals of C57BL/6J mice,Kunming mice,SD rats and Wistar rats were selected,respectively and divided into the experimental group and control group at average.The right eyes of the animals of the experimental group received the retro-orbital injection of 9 ml/kg FITC-dextran,and the right eyes of animals of the control group received PBS solution at the same volume and way.All the animals were sacrificed 10 seconds after injection and both eyes of each animal were obtained for retinal stretched preparation.The retrobulbar tissue and whole-mount retina were viewed under a fluorescence microscope.The use of the animals complied with Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Retinal blood vessels labeled by FITC-dextran could be observed in both eyes of C57BL/6J mice and Kunming mice to present with a green fluorescence in experimental group under a fluorescence microscope,but no any fluorescence-labeled retinal blood vessel was exhibited in the control mice.The retinal blood vessel could not be observed in all eyes of SD rats and Wistar rats after the injection of FITC-dextran both in the experimental group and the control group under a fluorescence microscope.The surrounding tissues of the right eyes of mice and rats dyed with green fluorescence of FITC-dextran in the experimental group,however,green fluorescence could not be seen in the surrounding tissues of the left eyes of mice and rats.Conclusions Retro-orbital injection of FITC-dextran is a suitable method of observing the retinal vessels of mouse but not rat.
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Nano-composite fluorescence with silica shell had been prepared by improving St(o)ber route. The precursor was obtained via chemical reaction of fluorescein isothiocyanate(FITC) and 3-aminopropyltrietho-)xysilane(APTEOS)). Then, the controlled hydrolysis and condensation of tetraethylorthosilicate(TEOS) leaded) to the formation of organic-inorganic nano-composites. The transmission electron microscope(TEM) showed that the nano-composites with a diameter of about 70 nm were uniform, spherical and mono-dipersed. The nano-composites fluorescence significantly reduced dye leaching and had strong fluorescence when they were by water for several times. Labeled bovine scrum albumin(BSA) with the nano-composites fluorescence showed green fluorescence measured by laser scanning confocal microscope.
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Em meados da década de 50 iniciou-se o desenvolvimento da citometria de fluxo, tecnologia que permite verificar características físico-químicas de células ou partículas suspensas em meio fluido. Esta tecnologia utiliza anticorpos monoclonais marcados com fluorocromos como ferramenta de investigação em diversas análises e necessita de controles isotípicos para definição da região negativa (background). Estes controles são constituídos por imunoglobulinas de mesmo isotipo e fluorocromo dos anticorpos testes, sendo o isotiocianato de fluoresceína (FITC) o marcador fluorescente mais utilizado na conjugação de anticorpos. Os controles isotípicos têm como função definir a fluorescência inespecífica (células negativas) e as regiões fluorescentes (células positivas). No presente estudo foi selecionado anticorpo monoclonal murino (AcMm) dirigido contra antígeno eritrocitário canino, produzido no Laboratório de Anticorpos Monoclonais do Hemocentro de Botucatu, o qual reage positivamente com hemácias de cães, mas nunca com leucócitos humanos, tendo, portanto, potencial utilidade como controle negativo em citometria de fluxo. A purificação do AcMm da subclasse IgG1 foi feita por cromatografia de afinidade em Proteína-A Sepharose, e o controle da purificação realizado por eletroforese em géis de ágarose e poliacrilamida (SDS-PAGE). A imunoglobulina purificada foi conjugada ao FITC e filtrado em coluna de Sephadex G-25 para separação das proteínas marcadas e não-marcadas. O AcMm conjugado foi testado contra hemácias de cães, e o êxito da conjugação comprovado por testes de fluorescência, sendo a mediana de positividade de 94,70. Frente a leucócitos humanos a mediana de positividade foi 0,03 contra 0,50 dos reagentes comerciais. Os testes estatísticos não-paramétricos de Wilcoxon e correlação de Spearman comprovaram a eficiência e validam o controle isotípico produzido em comparação aos reagentes comerciais testados.
It was during the 1950's that the development of flow cytometry started, technology that allow to measure physiochemical characteristics of cells or suspended particles in fluid. This technology uses monoclonal antibodies labeled by fluorochromes as investigation tool in several analysis and needs isotype controls to define the negative region (background). These controls are constituted by immunoglobulins of the same isotype and fluorochrome from test antibodies, being fluorescein isothiocyanate (FITC) the most fluorescent marker used in antibody conjugations. The isotype controls have the function to define the unspecific fluorescence (negative cells) and the fluorescent regions (positive cells). In this study was selected monoclonal antibody (mAb) against canine erythrocyte antigen, produced in the Monoclonal Antibodies Laboratory - Blood Center of Botucatu, which reacts positively with dog red blood cells, but never with human leukocytes, having therefore, utility potential as negative control in flow cytometry. The purification mAb of IgG1 subclass was made by affinity chromatography in Sepharose Protein-A and the purification control was performed by electrophoresis in ágarose and polyacrylamide gels (SDS-PAGE). The purified immunoglobulin was conjugated to FITC and after was filtered in Sephadex G25 column to separation of labeled and unlabeled proteins. The conjugated mAb was tested against dog red blood cells and the conjugation success was verified by fluorescence tests, being the median positivity of 94.70. To the human leucocytes the positivity median was 0.03 against 0.50 of the commercial reagents. The nonparametric statistical tests of Wilcoxon and the correlation Spearman showed the efficiency and validate the isotype control produced in relation to the tested commercial reagents.
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Fluoresceína-5-Isotiocianato , Sangue , Isotipos de Imunoglobulinas , Imunoglobulina G , Imunoglobulinas , Isotiocianatos , Eletroforese , Eletroforese em Gel de Poliacrilamida , Serviço de Hemoterapia , Citometria de Fluxo , Indicadores e Reagentes , Anticorpos , Anticorpos Monoclonais , AntígenosRESUMO
Objective To observe the effects of anti lipopolysaccharide (LPS) immunoglobulin Y (IgY) on LPS in vitro . Methods The direct in vitro neutralization of different concentrations of IgY and LPS was conducted for the detection of the remained amount of LPS to observe the dose effect relationship. The primary cultured human umbilical vein endothelial cells (HUVECs) were cultured for 48-72 h after addition of certain concentrations of fluorescein isothiocyanate LPS (FITC LPS) and IgY. Changes of the fluorescent intensity in HUVECs were observed at different time points. After addition of certain concentrations of LPS and IgY for incubation with HUVECs for 4, 8, and 12 h, the changes of lactate dehydrogenase (LDH) activity were measured. Results The dose effect relationship was satisfactory when the ratio was 16︰1. At 4, 8, and 12 h, fluorescent intensity was somewhat strong in FITC LPS group, but weak in IgY group, especially at 8 and 12 h. The LDH activity in medium increased obviously after incubation for 8 h in LPS group ( P