| The over development of the adipose tissue, leads to the decrease of meat quality in animal products,and to human, leads to obesity and related complications, such as diabetes, coronary artery disease, atherosclerosis, hypertension and hyperlipoidemia. Adipose tissue in human and animal body is mainly composed with adipocytes. Adipocytes derive from pluripotent stem cells, and the process of adipogenesis is described two phases. The first phase, known as determination, involves the commitment of a pluripotent stem cell to the preadipocyte lineage. The second phase, which is known as terminal differentiation, the preadipocyte takes on the characteristics of the mature adipocyte. Excessive body fat deposition is caused both by hypertrophy and hyperplasia of adipocytes. The molecular regulation of transcription factors in terminal differentiation of adipogenesis has been well characterized and elucidated over the past two decades than determination because most studies have used 3T3-L1 mouse preadipocyte lines. It is discovered that transcription factors that control the cascade of events leading to a fully differentiated adipocyte act downstream of complex signaling pathways that integrate signals from the surrounding microenvironment but not a independent events with the development of post-genomics and bioinformatics.Wnt/β-catenin signaling pathway is an important regulator of adipocyte differentiation which has established in recent years. In Wnt/β-catenin signalling pathway,β-catenin plays a central role as a transcriptional coactivator. The differentiation of 3T3-L1 was inhibited by Wnt/β-catenin signalling pathway, andβ-catenin and same Wnt proteins were highly expressed in the early stage of differentiation in 3T3-L1 in vitro. These results suggested that Wnt/β-catenin signalling pathway maybe also take important roles in determination phase of pluripotent stem cell to the adipocyte lineage. However, whether Wnt/β-catenin signaling pathway have characteristics of adipose-derived mesenchymal stem cells (ADSCs) remain elusived. Molecular mechanisms by which Wnt/β-catenin signaling alters stem cells fate have not been fully investigated.Being not only an important species to animal production industry, pig is also considered as a suitable model for the study of fat deposition and obesity due to its similarity with human in the trait of fat deposition. In this study, using porcine ADSCs, the time-spatial expression of Wnt/β-catenin signaling main factors, especiallyβ-catenin during adipogenic differentiation process were investigated with SQ RT-PCR and western Blotting technique. Porcine ADSCs were treated with LiCl, a activator of Wnt/β-catenin signaling, and the effects of LiCl on porcine ADSCs adipogenic differentiation and underlying molecular mechanism were detected by Oil red O staining, Oil red O extraction, indirect immunofluorescence, SQ RT-PCR and western Blotting technique. Porcineβ-catenin shRNA expression vector was constructed with retroviral plasmids pSuper-retro-puro, and effects of gene silencing ofβ-catenin were detected by Oil red O staining, Oil red O extraction, SQ RT-PCR and western Blotting after stable transfection of shβ-catenin RNA to porcine ADSCs.In addition, a porcine de-differentiated preadipocyte model were established by"ceiling culture"method. The differentiation of the peadipocyes were detected by Oil red O staining and RT-PCR after passage, and cell biology were studied as well in the present work.The main results were as follows:1. By stabilization of cytoplasmicβ-catenin by continuous treatment with LiCl, the adipogenic differentiation in porcine ADSCs was suppressed and the osteogenesis was stimulated. The expression of adipogenic transcription factors CCAAT/enhancer binding protein-α(C/EBPα) and peroxisome proliferator-activated receptor-γ(PPARγ) but not C/EBPβor C/EBPδwere decreased by LiCl.2. Theβ-catenin gene silencing enhanced the adipogenic differentiation of porcine ADSCs and rescued LiCl induced antiadipogenesis, and the expression of C/EBPαand PPARγwere increased after knockdown ofβ-catenin.3. Wnt/β-catenin signaling factors especially ofβ-catenin were highly expressed in porcine ADSCs and in the early stage of determination phase of adipogenic differentiation, but declined with the time of adipogenic differentiation.4. pSuper-retro-β-catenin shRNA vector were constructed, and stably transfectedβ-catenin shRNA to porcine ADSCs after selected by puromycin. 5. 95% porcine ADSCs express stem cells markers of CD44 and CD105, and have pluripotent such as an ability to differentiate into adipocytes, osteoblasts, and myocytes when induced in different medium.6. Mature adipocytes can de-differentiated to preadipocytes. Same as primary cultured stromal-vascular fraction cells, the preadipocytes can differentiated to mature adipocytes with ability of proliferation after passage culture, but purity and adipogenic differentiation is higher than stromal-vascular fraction cells. The preadipocytes has good heritage.Taken together, our study provides the molecular information on fate determination by Wnt/β-catenin signaling pathway in ADSCs. The adipogenic differentiation in porcine ADSCs was suppressed and the osteogenesis was stimulated when increasing ofβ-catenin. In contrast, theβ-catenin gene silencing enhanced the adipogenic differentiation. In addition, to establish a novel preadipocytes cell line with ability of proliferation and differentiation from adult mature adipocytes of pig.
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