AIM： To isolate and identify the biological characteristics of human colon cancer stem cells （SW1116 cells） and further study their proteome. METHODS： SW1116 cells were isolated and cultured with a serum-free medi...AIM： To isolate and identify the biological characteristics of human colon cancer stem cells （SW1116 cells） and further study their proteome. METHODS： SW1116 cells were isolated and cultured with a serum-free medium （SFM）. Sphere formation was assayed to observe the formation of colon cancer stem cell spheres. SW1116 cells were inoculated into a serum-containing medium for observing their differentiation characteristics. Proliferation curve and cross-resistance of SWl116 cells to different drugs were detected by MTT. Percentage of SP cells in SW1116 cells was detected with Hoechst33342 staining. Telomerase activity in SW1116cells was checked by polymerase chain reaction （PCR）-enzyme linked immunosorbent assay. Expressions of stem cell relevant genes and proteins were detected by reverse transcription-PCR and Western blot, respectively. Total protein was isolated from SW1116 cells by two-dimensional gel electrophoresis （2-DE） and differentially expressed proteins were identified by tandem mass spectrometry （MALDI-TOF/TOF）. RESULTS： The isolated SW1116 cells presented as spheroid and suspension growths in SFM with a strong self-renewal, proliferation, differentiation and drug-resistance ability. The percentage of SP cells in SW1116 cells was 38.9%. The SW1116 cells co-expressed the CD133 and CD29 proteins. The telomerase activity in SW1116 cells was increased. The expressions of different stem cell relevant genes and proteins were detected. The proteomic analysis showed that the 26 protein spots were differently expressed in SW1116 cells and 10 protein spots were identified as ubiquitin fusion- degradation l-like protein, nuclear chloride channel protein, tubulin 13, Raichu404X, stratifin, F-actin cap- ping protein α-1 subunit, eukaryotic translation elongation factor 1 delta isoform 2, hypothetical protein, glyceraldehyde-3-phosphate dehydrogenase and guanine nucleotide binding protein 13 polypeptide 2-like 1, respectively. CONCLUSION： SW1116 cells are biologically characterized by sel展开更多
Achyranthes bidentata Blume is widely used as a traditional Chinese medicine with the effects of nourishing the liver and kidneys and strengthening muscles and bones.In this work,a rapid and simple strategy was develo...Achyranthes bidentata Blume is widely used as a traditional Chinese medicine with the effects of nourishing the liver and kidneys and strengthening muscles and bones.In this work,a rapid and simple strategy was developed for characterizing phytoecdysteroids by ultra-high-performance liquid chromatography coupled with liner ion trap-Orbitrap mass spectrometry using electrospray ionization in the negative mode.As a result,47 phytoecdysteroids were unambiguously or tentatively characterized.Among them,seven known compounds were identified according to the reference standards along with molecular formula,retention time and fragmentation patterns,while others were mostly potential new compounds.Through targeted isolation,the structures of three new compounds were determined by NMR spectra,which were consistent with LC-MS characterization.The present study provides an efficient method to deeply characterize phytoecdysteroids.展开更多
基金Supported by Medical Guidance Project of Shanghai Science Committee (No. 10411961800)Youth Science Fund of Fudan University (No. 08FQ49)
文摘AIM： To isolate and identify the biological characteristics of human colon cancer stem cells （SW1116 cells） and further study their proteome. METHODS： SW1116 cells were isolated and cultured with a serum-free medium （SFM）. Sphere formation was assayed to observe the formation of colon cancer stem cell spheres. SW1116 cells were inoculated into a serum-containing medium for observing their differentiation characteristics. Proliferation curve and cross-resistance of SWl116 cells to different drugs were detected by MTT. Percentage of SP cells in SW1116 cells was detected with Hoechst33342 staining. Telomerase activity in SW1116cells was checked by polymerase chain reaction （PCR）-enzyme linked immunosorbent assay. Expressions of stem cell relevant genes and proteins were detected by reverse transcription-PCR and Western blot, respectively. Total protein was isolated from SW1116 cells by two-dimensional gel electrophoresis （2-DE） and differentially expressed proteins were identified by tandem mass spectrometry （MALDI-TOF/TOF）. RESULTS： The isolated SW1116 cells presented as spheroid and suspension growths in SFM with a strong self-renewal, proliferation, differentiation and drug-resistance ability. The percentage of SP cells in SW1116 cells was 38.9%. The SW1116 cells co-expressed the CD133 and CD29 proteins. The telomerase activity in SW1116 cells was increased. The expressions of different stem cell relevant genes and proteins were detected. The proteomic analysis showed that the 26 protein spots were differently expressed in SW1116 cells and 10 protein spots were identified as ubiquitin fusion- degradation l-like protein, nuclear chloride channel protein, tubulin 13, Raichu404X, stratifin, F-actin cap- ping protein α-1 subunit, eukaryotic translation elongation factor 1 delta isoform 2, hypothetical protein, glyceraldehyde-3-phosphate dehydrogenase and guanine nucleotide binding protein 13 polypeptide 2-like 1, respectively. CONCLUSION： SW1116 cells are biologically characterized by sel
基金supported by the National Key R&D Program of China(No.2019YFC1711000)the Qi-Huang Chief Scientist Program of National Administration of Traditional Chinese Medicine(2020).
文摘Achyranthes bidentata Blume is widely used as a traditional Chinese medicine with the effects of nourishing the liver and kidneys and strengthening muscles and bones.In this work,a rapid and simple strategy was developed for characterizing phytoecdysteroids by ultra-high-performance liquid chromatography coupled with liner ion trap-Orbitrap mass spectrometry using electrospray ionization in the negative mode.As a result,47 phytoecdysteroids were unambiguously or tentatively characterized.Among them,seven known compounds were identified according to the reference standards along with molecular formula,retention time and fragmentation patterns,while others were mostly potential new compounds.Through targeted isolation,the structures of three new compounds were determined by NMR spectra,which were consistent with LC-MS characterization.The present study provides an efficient method to deeply characterize phytoecdysteroids.
