ABSTRACT
Objective To explore a practical and feasible method for isolation,culture and identification of mouse bone marrow endothelial progenitor cells(EPCs).Methods Bone marrow-derived mononuclear cells isolated by density gradient centrifugation were cultured in endothelial cell growth medium-2 MV medium.Growth and morphological changes of the cells were observed under inverted microscopy.Cell proliferation was observed by cell counting kit-8 assay.Surface markers of the EPCs were detected by flow cytometry.Angiogenic tube formation was determined by Matrigel tube formation assay.Fluores cein isothiocyanat e-ulex europaeus agglutinin-1 (FITC-UEA-1) binding and Dil-Ac-LDL uptake capabilities were observed by fluorescent microscopy.Results In the early stage,the cells were round and spindle-shaped after induced culture for 4 days.After 7 days,the cells grew in colony arrangement and gradually increased in number.After 14 days,the cells were differently shaped,such as short shuttle and triangle.After 21 days,the typical "paving stone" appearance of the cells was observed.The cells were positive for endothelial markers in flow cytometry:CD34 + (84.3％),vascular endothelial growth factor receptor 2 + (74.1％),but CD45 + (4.04％).The cells were capable of forming capillary-like tubes,up-taking Dil-Ac-LDL and binding FITC-UEA-1 in Matrigels.Conclusions A reliable method for isolation,culture and identification of mouse bone marrow EPCs may be improved on the basis of previous experiences.Since the EPCs obtained by this method may be capable of good proliferation,large in number,and stable in biological characteristics,they can serve as ideal seed cells for related subsequent studies.
ABSTRACT
Objective: To establish an HPLC with charge aerosol detection (HPLC-CAD) method for the determination of kanamy-cin and kanamycin B in kanamycin sulfate injection. Methods: The samples were injected into a Boston Green ODS C18(250 mm× 4. 6 mm, 5 μm) column. The mobile phase was composed of 0. 2 mol·L-1trifluoroacetic acid aqueous solution-methanol (95: 5) , the flow rate was 1. 0 ml·min-1and the column temperature was maintained at 30 ℃. The nebulization temperature for the CAD was maintained at 55℃ and the gas pressure was 56. 4 psi. Results: The linear range of kanamycin was 0. 385-38. 500 μg·ml-1( r=0. 999 9), and the limit of detection was 0. 075μg·ml-1, the limit of quantitation was 0. 154 μg·ml-1, and the average recovery of the method was 100. 97% (n=9). The linear range of kanamycin B was 0. 374-37. 400 μg·ml-1(r=1. 000 0), and the limit of de-tection was 0. 075μg·ml-1, the limit of quantitation was 0. 150 μg·ml-1, and the average recovery of the method was 100. 44% (n=9). Conclusion: The method for the determination of kanamycin and kanamycin B by HPLC-CAD is simple and accurate, the limit of detection is lower than HPLC-ELSD, and the quality of kanamycin sulfate injection can be effectively controlled.
ABSTRACT
<p><b>OBJECTIVE</b>A RP-HPLC method was developed for simultaneous determination of bigelovin, ergolide and tomentosin in Inula hupehensis.</p><p><b>METHOD</b>An Agilent C18 column (4.6 mm x 250 mm, 5 microm) was used for separation at 40 degrees C. The mobile phase was acetonitrile-water, and the flow rate was 1.2 mL x min(-1). The detection wavelength was set at 210 nm.</p><p><b>RESULT</b>The method has good linearity in the ranges of 0.01792-0.1792 g x L(-1) (r =0.9999) for bigelovin, 0.0424-0.4240 g x L(-1) (r =0.9996) for ergolide, and 0.044 8-0.4480 g x L(-1) (r = 0.9996) for tomentosin. The average recoveries of bigelovin, ergolide, and tomentosin were 98.5%, 98.2%, 98.4%, with the RSD of 1. 3%, 1.3%, 1.7%, respectively. The results demonstrated that there was a significant difference in the contents of three sequterpene lactones among the tested Inulae Flos.</p><p><b>CONCLUSION</b>The results indicated that the present RP-HPLC method is simple, quick and accurate, and can be used for the quality control of I. hupehensis, especially for the authentication of Inulae Flos.</p>