To investigate the effect of hSCGF-alpha on human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs), we obtained hSCGF-alpha using genetic engineering, hSCGF-alpha gene was amplified from hUCMSCs cDNA using two-step PCR and was inserted into pET-28a(+) plasmid vector. Induced by IPTG at 20 degrees Celsius for 24 h, the fusion protein expressed in E. coli BL21 (DE3) was mainly existing in soluble form. The recombinant hSCGF-a was purified using NI-NTA affinity chromatography and the purity was up to 90%. The colony forming test revealed that combined use hSCGF-alpha and rmGM-CSF (recombinant murine GM-colony stimulating factor, rmGM-CSF) had granulocyte/macrophage (GM) promoting effects on murine bone marrow GM progenitor. In addition, the results indicated that hSCGF-alpha and rhGM-CSF had stimulatory effect on hUCMSCs and their synergetic effect was the strongest.
Cell Proliferation ; drug effects ; Cells, Cultured ; Cloning, Molecular ; Drug Synergism ; Escherichia coli ; genetics ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Stem Cell Factor ; biosynthesis ; genetics ; Umbilical Cord ; cytology

OBJECTIVE： To establish UPLC-Q- TOF-MS/MS fingerprint of Fritillaria thunbergii ， and to define its anti-inflammatory quality markers. METHODS ：The determination was performed on Eclipse Plus C 18 column with mobile phase consisted of methanol- 0.1％ formic acid solution （gradient elution ）at the flow rate of 0.4 mL/min. The column temperature was set at 30 ℃，and injection volume was 2 µL. The electrospray ion source was used to scan in the range of m/z 50-1 200 with positive and negative ion detection mode. UPLC-Q-TOF-MS/MS fingerprints of 10 batches of F. thunbergii from different habitats were established by using the Similarity Evaluation System of TCM Chromatographic Fingerprints （2004A edition ）. With ear swelling degree，the serum levels of MDA and NO as anti-inflammatory indexes ，the correlation between the relative area of common peaks in fingerprint and the anti-inflammatory indexes was analyzed by using bivariate correlation analysis and grey correlation analysis ， and the quality markers were screened and identified. RESULTS ：In positive and negative ion mode ，10 batches of F. thunbergii had 26 peaks and 10 peaks. Based on bivariate correlation analysis and grey correlation analysis ，nine quality markers related to anti-inflammatory effect were found ，which were identified as peiminine ，peimine，cyclobalamine，daucidin，polyphyllin Ⅴ， bitumen podophyllotoxin ，phytosterol，ent-kaur-15-en-17-ol，ent-17-norkauran-16-one. CONCLUSIONS ：Established UPLC-Q- TOF-MS/MS fingerprint can be used to evaluate the quality of F. thunbergii ；peiminine，peimine and cyclobalamine and so on may be the quality markers of anti-inflammatory effect of F. thunbergii .