Anti-breast cancer activity of ebracteolatain A / 第二军医大学学报

Objective To isolate and purify ebracteolatain A from the roots of Euphorbia ebracteolata Hayata, and to explore its anti-breast cancer activity. Methods Ebracteolatain A was isolated and purified from the roots of Euphorbia ebracteolata Hayata using reflux extraction, solvent extraction and absorption chromatography techniques. The chemical structure was identified by mass spectrometry and nuclear magnetic resonance spectrometry. The antibreast cancer activity of ebracteolatain A was determined by MTT assay in the breast cancer cell lines including SUM149 (triple-negative), MCF-7 (luminal A), ZR-75-1 (luminal B) and SK-BR-3 (HER2-positive). The effect of ebracteolatain A on cell cycle was determined by flow cytometry. A tumor model was established in nude mice by transplanting SUM149 cells, and the inhibitory effect of ebracteolatain A on breast cancer was evaluated. Results The ebracteolatain A was 3, 3’-diacetyl-2, 4’-dimethoxy-2’, 4, 6, 6’-tetrahydroxy-5’-methyl diphenylmethane. The half maximal inhibitory concentrations of ebracteolatain A on SUM149, MCF-7, ZR-75-1 and SK-BR-3 cells were 5.50, 6.16, 7.08 and 8.64 μmol/L, respectively. With the increase of drug concentration (2.5, 5, 10 μmol/L), the percentage of the cells at G0/G1 phase was decreased (P<0.05, P<0.01) and the percentage of the cells at S phase was increased (P<0.05, P<0.01) after treatment with ebracteolatain A for 12, 24 and 48 h. After intraperitoneal injection of ebracteolatain A 35 mg/kg, the inhibition rates of the tumor volume and mass in nude mice were 37.94% and 41.38%, respectively. Conclusion Ebracteolatain A can inhibit the proliferation of the four types of breast cancer cells and the growth of transplanted-SUM149 cell tumor on nude mice, which may be related to suppressing the transition of cell cycle from S phase to G2/M phase.

Expression patterns of PPARgamma2 in differentiation of mouse embryonic stem (ES) cells into adipocytes / 生物工程学报

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.

The labeling of 3T3-L1 preadipocyte cells with enhanced green fluorescent protein / 生物工程学报

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.