Migration And Differentiation Of Bone Marrow Derived Mesenchymal Stem Cells In High-Fat Diet-Induced Obese Rats

PART 1 PRE-ADIPOCYTE CULTURE IN VITRO【Objective】To optimize the method for culturing rat pre-adipocytes in vitro.【Subjects and Methods】Pre-adipocytes were cultured in vitro. Several modifications were made to improve Ng's original method. It included: time for digestion, centrifugation speed and frequency of infiltration. Cells were cultured in basic and differentiation media. Pre-adipocytes were detected by morphologic method, auxodrome study and stained with Oil red O for determining the cell identity.【Results】Using our modified method, primary cultured pre-adipocytes were harvested in greater amount compared with the original method. The primary cultured pre-adipocytes could differentiate into adipocytes under the induction of differentiation medium. The primary cultured pre-adipocytes obtained by our method had the characteristics of pre-adipocytes.【Conclusions】We have established an efficient primary pre-adipocyte culture method. Our modification method is better than the original one. PART 2 MIGRATION AND DIFFERENTIATION OF BONE MARROW DERIVED MESENCHYMAL STEM CELLS IN OBESE RATS FED HIGH-FAT DIET【Objective】To observe the migration of bone marrow derived mesenchymal stem cells (BM-MSCs) to adipose tissue and their differentiation to adipocytes in rats fed high-fat diet.【Subjects and Methods】A group of 33 male Sprague-Dawley rats (SD rats) at the age of 28 days were used. A dose of 2×106 cells/100 g green fluorescent protein-expressing MSCs (GFP-MSCs) was transplanted into rats via tale vein injection. After transplantation, 33 rats were randomly divided into standard chow (n=8) and high-fat diet (n=25) groups. The high-fat diet group were fed in the following method: 100% standard chow for 3 days, 25% high-fat diet + 75% standard chow for 2 days, 50% high-fat diet + 50% standard chow for 2 days,75% high-fat diet + 25% standard chow for the rest period of experiment. After 7 and 11 weeks of transplantation, 6 diet-induced obese (DIO) rats and 3 DIO rats, 8 control rats were killed and adipose tissues were separated, respectively. Fat tissue slides were made and detected under the laser confocal fluorescence microscope. The eGFP+ cells' identity was identified by adipocyte markers, i.e. PPAR-γ,C/EBP-α, perilipin and macrophage markers, c-fms,CD11b, and F4/80.【Results】Rats became obese after 6-7 weeks high-fat diet feeding. A number of eGFP+ cells were found in the obese (DIO) rats' fat pads, especially in the perirenal and mesentric fat pads. eGFP+ cells were found to be adipocytes morphologically under the microscope. For adipocyte markers, i.e. PPAR-γ, C/EBP-α, and perilipin, all were positive for eGFP+ cells under the laser confocal fluorescence microscope. Furthermore, the eGFP+ cells from fat pads of DIO rats were negative for macrophge markers detected, i.e. c-fms, CD11b, F4/80 under the laser confocal fluorescence microscope.