[The genetics of human monogenic obesity].

Obesity is a clinical syndrome caused by genetic and environmental factors and has a relatively high heretability. Seven genes, of whose mutations each can independently result in severe human obesity, have been cloned. Six of them are involved in the appetite controlling by the central nervous system, and one is related to the regulation of adipocyte differentiation. Investigations into the genetic basis of human obesity are important for understanding the mechanism of obesity formation and for design and screening of anti-obesity drugs.

[Establishing mouse embryonic stem cell line carrying a fluorescent undifferentiated marker].

To label mouse ES cells,a cell line derived from the inner cell mass of 3.5-day blastocysts,with enhanced green fluorescent protein (EGFP), the vector of pRex-1-EGFP was transferred into mouse ES cells by electroporation. The expressions of Rex-1 in undifferentiated and differentiated ES cells were detected by the microscopic observation of EGFP and by RT-PCR. The results showed that the EGFP gene was transferred into the mouse ES cell line, and the transfected cells in undifferentiated state showed high levels of EGFP expression. When the cells began to differentiate, the EGFP expressions were gradually reduced. A mouse ES cell line expressing EGFP under the control of Rex-1 gene promoter was generated. The cell line provides a powerful approach for the research of the process of mammalian development and for the screening of small molecules that can regulate this process.

[Adipocyte differentiation and its regulation].

Fat tissue and adipocytes have been exclusively investigated in the past two decades, especially in the last ten years, due to the following two reasons. Firstly, more and more studies showed that fat tissue is not only an organ for energy storage, but also an endocrine one that can secret many kinds of hormones or hormone-like peptides. Secondly, the established preadipocyte cell lines have been providing powerful tools for the in vitro research of adipocyte differentiation, because these immortal cell lines authentically represent, to a great extend, the in vivo situations of these cells and can be induced to differentiate into mature adipose cells with proper hormones. It has been demonstrated that there exists close relations between adipocyte differentiation and many physiological or pathological processes including saccharide and fat metabolism, energy balance, obesity, diabetes, hyperlipidemia and breast cancer. It is very important to make known the differentiation mechanisms of preadipocyte into adipocyte for understanding the above mentioned diseases and for screening anti-obesity and anti-diabetes drugs.

[Expression patterns of PPARgamma2 in differentiation of mouse embryonic stem (ES) cells into adipocytes].

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.

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.