To investigate the expression patterns of peroxisome proliferator activated receptor2 (PPARgamma2) gene in the differentiation of mouse embryonic stem (ES) cells into adipocytes, mouse ES cells were transfected with the vector of pPPARgamma2-promoter-luciferase, and PPARgamma2 expressions were analyzed by detecting luciferase activities and by detecting the protein expressions using western blotting. The results showed that the gene PPARgamma2 did not express in undifferentiated mouse ES cells and in embryoid bodies (EBs) within the first 2 days of differentiation induction after EB formation, and began to express from the third day of differentiation induction after EB formation to the finish of the differentiation. The gene's expression in differentiated adipocytes was much stronger than that in differentiating preadipocytes. In Conclusion our results reported for the first time the five-step expression patterns of the gene PPARgamma2 during the whole differentiation procedures from mouse ES cells into adipocytes via preadipocytes, and supported the previous studies that PPARgamma2 is a fat-specific gene that expresses only in developed and developing adipose tissues.
Adipocytes ; cytology ; Animals ; Cell Differentiation ; genetics ; Cells, Cultured ; Electroporation ; Embryonic Stem Cells ; cytology ; metabolism ; Mice ; PPAR gamma ; biosynthesis ; genetics ; Promoter Regions, Genetic ; genetics ; Transfection

A cell model is desired for adipocyte differentiation investigation and for high-throughput screening of anti-obesity and anti-diabetes molecules from chemical resources due to the world wide epidemic of obesity and diabetes. In order to establish such a cell model, a plasmid of pPPARgamma2-promoter-EGFP was constructed by inserting a 660bp sequence of mouse PPARgamma2 promoter into the Ase I and Kpn I sites of pEGFP-N3 and transferred into 3T3-L1 preadipocyte cells. The cells were induced to differentiate and the expression of PPARgamma2 was detected by the microscopic observation of EGFP and by RT-PCR assays. The results showed that the EGFP gene expression patterns were similar to that of pPPARgamma2's, which indicated that the EGFP gene was transferred into the mouse 3T3-L1 preadipocyte cells, and its expression was under the control of pPPARgamma2 promoter. RT-PCR assays showed that the EGFP expression authentically represented the stable expression of PPARgamma2. In conclusion, a preadipocyte cell line expressing EGFP under the control of the promoter of adipocyte-specific expression gene PPARgamma2 was generated. The cell line provides a powerful approach for the research of adipocyte differentiation and for the high-throughput screening of anti-obesity and anti-diabetes chemicals.
3T3-L1 Cells ; Adipocytes ; cytology ; Animals ; Cell Differentiation ; Green Fluorescent Proteins ; genetics ; Mice ; PPAR gamma ; genetics ; Promoter Regions, Genetic ; Stem Cells ; cytology

OBJECTIVETo observe the effect of tetrandrine on activity of collagenase derived from human hypertrophic scar for the sake of clarifying the mechanism as tetrandrine acting on scar.

METHODSThe experimental concentration was controlled below that of cell proliferation inhibited, SDS-PAGE electrophoresis was adopted to separate collagenase from extracellular matrix, and then activated by trypsin analyzed the activity of collagenase with density scanning apparatus. At the same time quantity of extracellular collagen was measured using improved chloraseptine T oxidizing assay, moreover analyzed correlation between activity of collagenase and quantity of extracellular collagen.

RESULTSIn the concentration below the lever of inhibiting fibroblast proliferation, the total activity of collagenase could be significantly increased by tetrandrine with dosage-dependence associated with quantity of extracellular collagen reduced, which was much greater than that of triamcinolone.

CONCLUSIONIncreasing activity of collagenase on degradation of collagen even in a lower concentration was one of the mechanisms of tetrandrine treating hypertrophic scar.

Benzylisoquinolines ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Collagenases ; metabolism ; Fibroblasts ; cytology ; Humans

OBJECTIVE: To study the chemical constituents from the roots of Curcuma longa. METHODS: The structures of the compounds were elucidated based on extensive spectral analysis, including 1D and 2D NMR, MS, UV, and CD analysis. RESULTS: Two new sesquiterpene compounds (1S,2R,5R,7S,8R)-2,8-epoxy-5-hydroxybisabola-3,10-dioen-9-one ( 1), (1R,2R,5R,7S,8R)-2,8-epoxy-5-hydroxybisabola-3,10-dioen-9-one ( 2), and a new natural product 6-(4-Hydroxymethylphenyl)-2-methyl-hept-2-ene-4-one ( 3) together with three known compounds ar-turmerone ( 4), 2-methyl-6-(4-hydroxyphenyl-3-methyl)-2-hepten-4-one ( 5) and 2-methyl-6-(4-hydroxyphenyl)-2-hepten-4-one ( 6) were isolated from C. longa root extract with 95% ethanol. CONCLUSION In the study, three new compounds were isolated from C. longa, and their absolute configurations were determined.