We developed a high-efficiency microfluidic chip for extracting exosomes from human plasma. We collected peripheral blood from normal human, designed and fabricated a microfluidic chip based on nanoporous membrane and agarose gel electrophoresis to isolate exosomes. The extracted exosomes were characterized by transmission electron microscopy, nanosight and Western blotting, the morphology, concentration and particle size of exosomes were identified and analyzed. Meanwhile, we used ultracentrifugation and microfluidic chip to isolate exosomes separately. The particle size and concentration of the exosomes extracted by two methods were compared and analyzed, and their respective extraction efficiency was discussed. Finally, the expression level of miRNA-21 in exosomes was analyzed by RT-PCR. The microfluidic chip isolated (in 1 hour) high-purity exosomes with size ranging from 30-200 nm directly from human plasma, allowing downstream exosomal miRNA analysis. By comparing with ultracentrifugation, the isolation yield of microfluidic chip was 3.80 times higher than ultracentrifugation when the volume of plasma sample less than 100 μL. The optimized parameters for exosome isolation by gel electrophoresis microfluidic chip were: voltage: 100 V; concentration of agarose gel: 1.0%; flow rate of injection pump: 0.1 mL/h. The gel electrophoresis microfluidic chips could rapidly and efficiently isolate the exosomes, showing great potential in the research of exosomes and cancer biomarkers.
Exosomes ; Humans ; MicroRNAs/genetics* ; Microfluidics ; Plasma ; Ultracentrifugation

The measurement and analysis of sedimentation equilibrium provides one of the most powerful and widely applicable methods for the characterization of reversible associations of macromolecules in solution. Recent developments in instrumentation, experimental design, and data analysis have substantially broadened the range of systems to which this technique may be applied, simplified its application, and reduced the cost of acquiring analytical capability.
Animal ; Human ; Kinetics ; Macromolecular Systems* ; Molecular Weight ; Proteins/chemistry ; Proteins/analysis ; Solutions ; Ultracentrifugation/methods ; Ultracentrifugation/instrumentation*

OBJECTIVE: To investigate the role of exosome in mediating bortezomib (Btz) resistance in multiple myeloma cells and explore the underlying mechanisms. METHODS: Peripheral blood samples were collected from 15 patients with multiple myeloma with Btz tolerance, and serum exosomes were isolated by ultracentrifugation and identified with electron microscopy, NTA and Western blotting. cultured multiple myeloma cells were treated with gradient concentrations of Btz to determine the optimal drug concentration for subsequent experiment. The cells were pretreated with different concentrations of exosomes, and their sensitivity to BTZ was assessed using MTS assay. We searched the exosome database Exocarta and used STRING to generate the network map and the protein interaction graph. RESULTS: The diameters of the vesicles isolated from the peripheral blood of the patients were mostly below 200 nm with a mean particle size of 153 nm and a mode of 140.1 nm. The results of Western blotting showed that the isolated exosomes expressed the marker proteins CD63, Tsg101 and Alix. In cultured multiple myeloma cells, pretreatment with exosomes resulted in a decreased sensitivity of the cells to bortezomib, and longer treatment durations and higher exosome concentrations consistently enhanced the resistance of the cells to the same Btz concentration. Analysis of the Exocarta database identified human serum exosomal proteins ABCB1, ABCB4, PDCD6IP, and EGFR, among which EGFR served as a network node. CONCLUSIONS Exosome within a specific concentration range may serve as a signal carrier to mediate the resistance of multiple myeloma cells to Btz. EGFR likely plays a key role to promote exosome-mediated Btz resistance in myeloma cells.
Bortezomib ; Drug Resistance, Neoplasm ; Exosomes ; Humans ; Multiple Myeloma ; Ultracentrifugation

BACKGROUND: The size reduction of low density lipoproteins(LDL) particles has been found associated with coronary artery disease(CAD), but the analysis of LDL subfractions is time consuming(over 24 hours) and technically demanding such as density gradient ultracentrifugation or gradient polyacrylamide gel electrophoresis(PAGE). In this study, the association between LDL subfractions and CAD was investigated using the new method for the separation of LDL subfractions, commercially available continuous disc PAGE. METHODS: LDL subfraction profiles were investigated in two groups(97 patients with CAD proven angiographically and 109 healthy controls) using Lipoprint LDL System(Quantimetrix Co., Redondo Beach, CA, USA). Seven LDL subfractions(from LDL-0 to LDL-6) can be identified with a specific electrophoretic mobility(Rf) relative to the HDL fraction. The relative percentage of the area under the curve(AUC) of each LDL band was multiplied by its band number and the sum of the relative AUC of all LDL bands percent was calculated to produce a final LDL score. RESULTS: Mean LDL score was significantly higher in CAD patients than in controls(2.09+/-0.77 and 1.01+/-0.38, respectively, P<0.0001). There was a significant association between the polydispersity of LDL subfractions and the presence of CAD(P=0.01). LDL score was highly correlated with triglyceride(r=0.330, P=0.0001). LDL score was the significant discriminators between control and CAD groups(F=168.3, P=0.0001). There was no significant association between LDL score and the severity of CAD. CONCLUSIONS: Lipoprint LDL System is an easy and quick method for routine assessment of LDL subfractions. LDL score was the best individual discriminant risk factor for CAD.
Area Under Curve ; Coronary Artery Disease* ; Coronary Vessels* ; Humans ; Lipoproteins* ; Risk Factors ; Triglycerides ; Ultracentrifugation

PURPOSE: Nitrocellulose membrane–based filtration system (NCFS) is widely used for protein concentration. In this study, we applied NCFS for production of virus-like particle (VLP) as a vaccine candidate and evaluated yield property and immunogenicity. MATERIALS AND METHODS: Influenza VLPs were generated by baculovirus-insect cell protein expression system. NCFS and sucrose gradient ultracentrifugation were used for purification of VLP. Immunogenicity of VLP was evaluated by animal experiment. RESULTS: Influenza VLPs expressing hemagglutinin (HA) and neuraminidase proteins derived from highly pathogenic influenza virus (H5N8) were effectively produced and purified by NCFS. HA activity of VLP which correlated with antigenicity was well conserved during multiple purification steps. This NCFS based purified VLPs induced influenza virus–specific antibody responses. CONCLUSION: Our results indicate that the influenza VLP vaccine could be prepared by NCFS without loss of immunogenicity and elicit antigen-specific immune responses.
Animal Experimentation ; Antibody Formation ; Baculoviridae ; Collodion* ; Filtration* ; Hemagglutinins ; Influenza, Human* ; Membranes* ; Neuraminidase ; Orthomyxoviridae ; Sucrose ; Ultracentrifugation ; Vaccines

PURPOSE: There has been a renewal of interest in Macrophage migration inhibitory factor (MIF), especially correlation in pathogenesis of sepsis by many infectious diseases and in regulation of host inflammatory and immune response. We developed immunoradiometric assay (IRMA) to determine serum human MIF concentration. MATERIALS AND METHODS: The IRMA system utilizes solid phase bound monoclonal anti-recombinant human MIF (rhMIF) antibody as a capture antibody, biotinylated polyclonal anti-rhMIF antibody as a detector antibody. We applied with rhMIF that concentration of standard solutions increased from 0 ng/ml to 100 ng/ml. We used 125I-streptavidin (SA) as radiotracer to determination of rhMIF concentration. Streptavidin was labeled with 125I by Chloramine-T method and 125I-SA was purified by ultracentrifugation. 125I-SA stability was evaluated by ITLC analysis at 4 degrees C and room temperatures until 60days. To validate IRMA system for MIF, we experimented intra-assay and inter-assay coefficients of variation, recovery test and dilution test. RESULTS: Radiolabeling yield of 125I-SA was 87% and purified 125I-SA retained above 99% radiochemical purity. 125I-SA showed above 93% stability in 4 degrees C until 60days that it is good for immunoradiometric assay as radiotracer. Plotted standard dose response curve showed that increased concentration of rhMIF linearly correlated (R2=0.99) with bound radioactivity of 125I-SA. The highest intra- and inter-assay coefficients of variation were 5.5% and 7.6%, respectively. The average of recovery of MIF in samples was 102%. In dilution test, linear response curves were obtained (R2=0.97). CONCLUSION: Radioimmunoassay using 125I-SA as radiotracer thought to be useful for the determination of serum MIF concentration, and further, its data will be used to evaluate the correlation between clinical significance and serum MIF concentration in patients with various inflammatory diseases.
Communicable Diseases ; Humans ; Immunoradiometric Assay ; Macrophages* ; Radioactivity ; Radioimmunoassay* ; Sepsis ; Streptavidin ; Ultracentrifugation

BACKGROUND: Elevated level of low density lipoprotein-cholesterol (LDL-C) is one of the major risk factors for the development of coronary heart disease. Direct LDL-C determination method by immunoseparation (DLDL-C) recently developed is claimed not to be influenced by food ingestion. We re-evaluated the effects of diet and storage conditions for this method. METHODS: Samples were collected from thirty-two medical college students before and after meal to study the effects of diet on this method. We compared the difference of LDL-C of filtered samples between refrigerated and frozen state. We also compared direct and indirect calculated measurements of LDL-C with ultracentrifugal beta-quantification (BQLDL-C) method. RESULTS: Morning 2-hour-postprandial specimen can be acceptable with no minimal significant bias, but afternoon 2-hour or 4-hour-postprandial specimen cannot be recommended due to significant negative bias (8.6-9.6%). Storage of filtered samples showed no significant difference between frozen and refrigerated state. Calculated LDL-C when triglyceride level is more than 400 mg/dL was not reliable due to large proportional and constant bias. In contrast, DLDL-C showed good accuracy comparing with BQLDL-C (y=0.909x+3.3, r=0.869, n=9, x=BQLDL-C, y=DLDL-C). CONCLUSION: In conclusion, morning two-hour postprandial specimens can be acceptable for DLDL-C, but afternoon postprandial specimens may not be recommended due to significant negative bias. DLDL-C seems to be reliable and useful especially for hypertriglyceridemic patients or follow-up cases of hypercholesterolemia with normal triglyceride or HDL-C levels.
Bias (Epidemiology) ; Cholesterol, LDL ; Coronary Disease ; Diet ; Eating* ; Fasting ; Humans ; Hypercholesterolemia ; Meals ; Risk Factors ; Triglycerides ; Ultracentrifugation

OBJECTIVEApplying for the activity of enzyme in vitro,the research optimized the best preparation procedure for the anticoagulated blood region from Lumbricus.

METHODAll through our experiment, the content of protein and theactivity of enzyme were examined. The extraction process, the refining technology, concentration processes of Lumbricus were optimized with single factor checking and orthogonal design method.

RESULTAt 37 degrees C, the coarse powder of Lumbricus soaking with 15 fold of 0.9% sodium chloride and ultrasonic extracting 40 minites for three times was the best ultrasonic extraction. Utrafiltration membrane with molecular weights of 30 x 10(3) for refining and 10 x 10(3) for concentrating were selected.

CONCLUSIONUltrasonic extraction and membrane separation technology, to well improve the effect of purification for the anticoagulant site of Lumbricus, is conducive to further study.

Animals ; Anticoagulants ; chemistry ; isolation & purification ; Drug Compounding ; methods ; Oligochaeta ; chemistry ; enzymology ; Temperature ; Ultracentrifugation ; Ultrasonics

OBJECTIVETo present an improved method to obtain pure, viable, freshly isolated hepatic stellate cells.

METHODSAdult male SD rats were used. All procedures were performed with the animals under sodium pentobarbital anesthesia. Three days after the single intravenous administration of 1 ml liposome-encapsulated CL2MDP, which has selective cytotoxicity of Kupffer cells, livers were perfused with D-Hank's solution containing 100 U/ml heparin for 10 to 15 minutes, and then with 0.05% collagenase dissolved in D-Hank's solution for 25 to 30 minutes. The liver was then gently homogenized and further incubated in 0.025% collagenase, and 0.005% DNAase I for 30 minutes at 37 degrees C under constant stirring. This suspension was filtered through stainless steel gauze and centrifuged for 2 minutes at 50 x g to remove parenchymal cells. Sinusoidal cells in the supernatant were recovered by centrifugation for 10 minutes at 300 x g. The cells were resuspended in the presence of 28.7% Nycodenz stock solution. The final concentration of Nycodenz at this stage was 11.5%. Following centrifugation for 17 minutes at 1400 x g, The cells at the top of this Nycodenz solution were collected. Cells were resuspended in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, The cells were seeded in 50 ml culture flask at a density of 500,000 cells/ml, The cell viability was determined by trypan blue exclusion staining, the purity of hepatic stellate cells was identified by the expression of Desmin using immunocytochemistry method. Endogenous peroxidase staining was used to detect Kupffer cells.

RESULTSThe yield rate of hepatic stellate cells was 3 x 10(7) per rat, the cell viability was more than 95%, the desmin positive cell rate was 90%, no endogenous peroxidase positive cells were detected.

CONCLUSIONThe method for the isolation of hepatic stellate cells was developed without Kupffer cells confusion. The availability of highly purified stellate cells will facilitate the investigation of their functions in primary culture.

Animals ; Cell Culture Techniques ; Cell Separation ; methods ; Kupffer Cells ; cytology ; Liver ; cytology ; Male ; Rats ; Rats, Sprague-Dawley ; Ultracentrifugation ; methods

Band 3, the predominant 95,000 dalton anion transport protein, is the major intrinsic glycoprotein of the human erythrocyte membrane. This anion carrier exists as a dimer and binds the cytoskeletons such as spectrin, ankyrin and actin. And the liposomes are vesicular structures which form spontaneously upon hydration of phospholipids. These artificial lipid vesicles have been investigated as model of the biological membranes and as a mean of improving the delivery of nucleic acids, drugs, proteins and biological substances to specific target tissues and cells. In this study, we were purified Band 3 from the human erythrocyte membrane (ghost) was prepared by hemolysis of intact human erythrocyte with weak alkali-hypotonic solution. Band 6 was removed from ghost by extracting with solution of an ionic strength of 0.15. Band 3 and Band 4 were solubilized selectively by extracting Band 6-depleted ghosts with Triton X-100 under nondenaturing conditions. Band 3 was then purified from Triton X-100 extract treated with p-chloromercuribenzoate by sucrose density gradient ultracentrifugation. This purified Band 3 was incorporated into liposomes prepared by reverse-phase evaporation. Phosphatidyl L-serine and cholesterol (1:1 molar ratio) were dissolved in chloroform and the chloroform was removed by rotator evaporation under reduced pressure. Band 3 solution without Triton X-100 was introduced into a mixture of lipids and diethylether. Diethylether was subsequently removed by evaporation. This purified Band 3 and its incorporation into liposomes were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Actins ; Ankyrins ; Chloroform ; Cholesterol ; Cytoskeleton ; Electrophoresis ; Erythrocyte Membrane* ; Erythrocytes* ; Glycoproteins ; Hemolysis ; Humans* ; Liposomes* ; Membranes ; Molar ; Nucleic Acids ; Octoxynol ; Osmolar Concentration ; Phospholipids ; Serine ; Sodium ; Spectrin ; Sucrose ; Ultracentrifugation