ABSTRACT
An in vitro detection method of the gastrointestinal absorption of Pilose Antler protein was established for mixed protein activity. Five bands of protein with molecular weight of 17.8-160 kD derived from the Pilose Antler were extracted and sufficiently labeled with FITC (FITC-PE). The stability and variation of FITC-PE in gastrointestinal circumstances were detected by native polyacrylamide gel electrophoresis and confocal laser scanning microscope. Results showed that the main component of FITC-PE kept invariant after being reacted with artificial gastric fluid and artificial intestinal fluid. The fluorescence signal was detected 20 min after administration in the valgus intestinal purse experiment, and three kinds of protein, with molecular weight of 45, 25, and 17.8 kD, were detected in the mixture of absorbent protein. The research laid the foundation for the further in vivo study of Pilose Antler protein. Meanwhile, it would be an in vitro screening method for the absorption, distribution and metabolism of mixed protein from traditional Chinese medicine.
ABSTRACT
<p><b>OBJECTIVE</b>To establish a new, real time, dynamic and direct optical detection method for mast cell degranulation caused by anaphylactoid reaction.</p><p><b>METHOD</b>A CD63-GFP plasmid was constructed and introduced steadily into rat basophilic leukemia (RBL-2H3) cells. The movements of CD63-GFP, which was located on both the granule membranes and the plasma membranes of RBL cells stimulated by Compound 48/80, were studied by confocal laser scanning microscope (CLSM) and total internal reflection fluorescence microscope (TIRFM) both inside and on the surface of living RBL-2H3 cells.</p><p><b>RESULT</b>Before antigen stimulation, most granules with CD63-GFP hardly moved in RBL cells. However, after antigen stimulation, the granules moved dramatically. They reached the plasma membranes in a few minutes and fused with them instantaneously. The velocity of the granule movement toward the plasma membranes on antigen stimulation was calculated to be 0.05 micron x s(-1).</p><p><b>CONCLUSION</b>Analysis of the movement of each granule provided a new insight into the elementary process of degranulation. The method is rapid, sensitive and reliable, which could be used as a new detection method for anaphylactoid reaction in vitro.</p>
Subject(s)
Animals , Rats , Anaphylaxis , Diagnosis , Allergy and Immunology , Metabolism , Antigens, CD , Genetics , Cell Degranulation , Cell Line, Tumor , Cell Movement , Mast Cells , Cell Biology , Allergy and Immunology , Microscopy, Confocal , Microscopy, Fluorescence , Platelet Membrane Glycoproteins , Genetics , Tetraspanin 30 , Time FactorsABSTRACT
Aim To establish a new,multi-parameter,real time and qualitative cell migration evaluation method.Methods Lung cancer cell line A549 was cultured on the glass bottom dish.After treated with different dosages of berberine or Rg3 for 24 hours,several scratching lines at the same dimension were made and observed by Living Cell Working Station.8 observation areas were selected randomly and imaged continuously for 24 hours.Transferred Area(TA),Transferred Distance(TD),Single Cell Transferred Speed(SCTS)and the Cell Division Number among defined area(CDN)were analyzed after getting sequence images.Results The focus stage and the incubation system were sufficient to keep cell proliferation and made it possible for long term observation.Berberine at 25 μmol·L~(-1) and 12.5 μmol·L~(-1) could inhibit the migration of A549(P<0.01).The analysis results of TA,TD,SCTS and CDN were basically coincident.Rg3 at 0.1 mmol·L~(-1) could inhibit SCTS and promote CDN in 6,12 and 24 h(P<0.01),while decrease both TA and TD in 24 hs.Conclusion The method is sensitive,rapid and simple to be applied in the research of tumor metastasis,wound healing and inflammatory response with real time,in-situ and multi-parameters.
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Here we reviewed the applications of laser scanning confocal microscope in biomedical field. The reformation and improvement based on this technique were also summarized. After analyzing the defects, we highlighted its potential applications in the future.