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Cocrystal separation technology is a technology that utilizes coformers to selectively form cocrystals with target compounds and separate them from mixed systems. Our study used puerarin (PUE), daidzein (DDZ), and genistein (GEN) as model drugs, which have similar structures and are the main isoflavones in Pueraria lobata root. The separation and purification processes in the modern traditional Chinese medicine (TCM) of these three components use conventional column chromatography, recrystallization, and other technologies, which have the issues of lengthy separation cycles, high solvent consumption, and inefficient preparation. Different with existing separation technology, our team used the early-found cocrystal separation method to design a step-by-step extraction and separation experiment of GEN-PUE-DDZ ternary mixture. Caffeine and L-proline were added to the mixed system in turn, GEN-caffeine cocrystal and PUE-proline cocrystal were prepared by suspension method. The cocrystals precipitated out of the solution. The purities of the GEN-caffeine cocrystal and the PUE-proline cocrystal could achieve 93% (the purity of GEN) and 99% (the purity of PUE). Besides, the purity of DDZ could also be increased by 6.76 times. This study proposed a simple operating, low cost and wide application range separation method different from the traditional separation method and realized the separation of structurally similar chemical components in TCM, laying a foundation for the application of cocrystal technology in the separation and refining of TCM.
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OBJECTIVE@#To construct a magnetic and catalytic hairpin assembly-based platform for detection of dual membrane proteins on exosomes.@*METHODS@#Exosomes in supernatant of breast cancer MDA-MB-231 cell culture were separated, purified and characterized. Super-resolution imaging and Western blotting were performed to confirm the expression of the membrane protein CD63 on the exosomes. Polyacrylamide gel electrophoresis was used to verify the combination of Apt-T and exosomes. Fluorescence experiments were carried out to test the feasibility of CHA nucleic acid sequence, optimize the reaction conditions, and determine the specificity of the detection platform.@*RESULTS@#Super-resolution imaging and Western blotting showed that breast cancer MDA-MB-231 cell-derived exosomes expressed abundant membrane protein CD63. Polyacrylamide gel electrophoresis indicated that Apt-T could recognize and bind to exosomes. The results of specificity test showed that the signal-to-noise ratio of the detection platform was 1.10±0.01 for detecting normal human breast epithelial cell-derived exosomes, and was 2.09±0.08 for breast cancer cell-derived exosomes.@*CONCLUSIONS@#Magnetic and catalytic hairpin assembly-based detection platform allows simultaneous detection of two membrane proteins expressed on exosomes and identification of the expressions of membrane proteins on exosomes from different sources.
Subject(s)
Humans , Blotting, Western , Exosomes , Magnetic Phenomena , Membrane Proteins , Sensitivity and SpecificityABSTRACT
Objective To explore a new method for the separation of human pancreatic stellate cells.Methods Single-cell suspension of normal pancreatic tissue and pancreatic cancer tissue was prepared by gentle MACSTM tissue processor-constant temperature shaking digestion.Human pancreatic stellate cells of quiescent and activated state were isolated by density gradient centrifugation.Results A new type of isolation method could obtain about (2.6 ± 0.7) × 106 quiescent pancreatic stellate cells in 1 g of human normal pancreatic tissue,with a viability of about 90.0％.The morphology of the cells were conformed to the representative for the quiescent state characteristics and transient blue-green autofluorescence was observed at the 328 nm excitation wavelength;1 g of human pancreatic cancer was able to obtain approximately (4.1 ± 1.1) × 106 activated PSCs with a viability of 92.0％,and all of the activated cells expressed α-SMA vimentin,FSP-1 and other characteristic markers.Conclusions The new separation method of this experiment is suitable for both human resting and activated human pancreatic stellate cells.At the same time,the purity is high and the separation time is greatly shortened,which is worth promoting.
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Objective@#To explore methods using modified tissue enzymatic separations for culturing primary hDPSCs in vitro and further identify the cells produced. @*Methods @#Primary hDPSCs were cultured using the modified tissue enzymatic separation method, and cells were identified by morphology, cell surface markers, and differentiation potential and evaluated using flow cytometry and growth curves.@*Results @#The hDPSCs were successfully isolated using the modified tissue enzymatic separation method. The morphology of these cells was similar to that of fibroblasts and mesenchymal stem cells, and the growth curve was "S" -type. The results of cell phenotype analysis indicated that the cells were positive for surface markers of mesenchymal stem cells, including CD29, CD44, and CD90, and negative for markers of hematopoietic stem cells, including CD34, CD45, and CD106. The cells were capable of differentiating into multiple cell types.@*Conclusion @# The modified tissue enzymatic separation method can successful be used to culture primary hDPSCs in vitro.
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Objective: To evaluate the optimal separation and purification processes of water extract, the analytic techniques of particle size analysis, powder fluidity testing, and scanning electronic microscope were adopted to compare the influence factor of different separation and purification techniques on microscopic preparation characteristic of intermediate product prepared from water extract of traditional Chinese medicine (TCM) formula purification. Methods: Taking Gubi Granules (GG) which have established the production technology and quality standards in the previous study as an example, three common techniques used for water extract of TCM formula purification, including ethanol precipitation, column chromatogram of macroporous resin, and membrane separation, were applied to preparing the intermediate of GG. Results: Through the comprehensive analysis of fluidity, adhesive property, compressibility, permeability, particle microstructure and particle size distribution, it was found that membrane separation could obtain intermediates with better performance, which was conducive to the subsequent granulation, tabletting, and other process smoothly. The operating conditions for liquid concentration were 0.05 g crude drug/mL, 30 ℃ liquid temperature, 0.15 MPa pressure and 5 m/s flow rate. Conclusion: The different spray dried powders varied greatly. The membrane separation method of 0.2 μm Al2O3 ceramic was selected as the optimal process for separating and purifying of water extract of GG by analyzing the influencing factors of the pharmaceutical properties.
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Objective To explore a separation and culture method of human adipose-derived stem cells(ADSCs) suitable for the clinical application .Methods The non-enzymatic method and the collagenase digestion method were adopted to isolate and culture the cells from the human adipose tissue in the individuals with liposuction .The characteristics of isolated mesenchymal stem cells were comparatively analyzed .Results The required time in the non-enzymatic method was one third of that in the collagenase di-gestion method and the cellular morphology ,reproductive capacity ,immunophenotype and differentiation potential of the isolated cells were consistent to those isolated by the collagenase digestion method .Conclusion The no-enzymatic method may isolate and culture ADSCs from the adipose tissue in the individual with liposuction ,which is a safety and reliable isolation and culture method of human adipose tissue-derived stem cells suitable for clinical application .
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In the present study, gelatin microspheres containing ofloxacin were prepared by coacervation phase separation method and characterized by optical microscopy and scanning electron microscopy. The microspheres were analyzed for drug entrapment, bulk density, angle of repose, particle size and In-vitro release pattern. The effect of process variables on microsphere size was studied and based on these preliminary studies, different batches of microspheres were prepared by altering the drug: polymer ratio and cross-linking with glutaraldehyde. The size of microspheres was in range of 42- 45μm. They were spherical in shape as evidenced by photomicrographs and scanning electron microscopy. The percent drug entrapment was in the range of 78-90 % and they could sustain drug release over a period of 8 hrs.
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A symmetric approach, using external rhinoplasty, is presented to aid the plastic surgeon in obtaining improved aesthetic and functional results in patients with postoperative nasal deformities. The external approach yields a full visualization of the underlying nasal framework and intraoperative evaluation of the deformities to be corrected subsequently. The nasal septal cartilage is unequivocally one of the best graft sources for reconstruction of the dorsum, columella or tip. It has fairly even surface and pliability in carving and shaping the graft. The graft can be obtained during the surgery with less morbidity and prepared easily for need of the shape. The only real disadvantage is the limited amount of cartilage that can be obtained from the septum. The dorsal and caudal rims, one or more cm in width, of the nasal septum should not be disturbed to maintain the nasal frame during harvesting the septal graft. Authors invented novel instruments, J & D knife and Flat (Spatula) suction tip, and have employed the devices for harvesting the septal cartilage. We were unable to gain enough amount of the cartilage by using a swivel knife or cartilage scissors. The septal cartilage can be resected as much as needed with newly invented instruments which facilitate a separation(method) technique.