High-Fat Diet-Induced Obese Rats And Migration And Differentiation Of Mesenchymal Stem Cells In Vivo

PART ONE CELL CULTURE IN VITRO【Objective】To optimize the methods for culturing rat adipocytes and preadipocytes in vitro.【Subjects and Methods】Adopocytes and preadipocytes were cultured in vitro. Modifications including: adding adipocyte into the wells, centrifugation speed, the type of enzymes, additives and time for digestion. Modified ceiling culture method was used to culture adipocytes. Fat tissues were digested by collagenase I for 60 - 90 minutes. Then the digested tissue was centrifuged for 5 minutes at 250 g. After the cells were washed and centrifuged for several times, the cells were resuspended by DMEM/F12 containing 20% new-born bovine calf serum and inoculated into a six-well plate. When the cells had become firmly attached, the culture medium was added slowly. Enzyme-digesting methods were used to culture preadipocytes in vitro. After digestion, the cells suspension were centrifuged at 800g for 5 minutes. The cells were washed and centrifuged for several times and were incubated in a culture flask. The floating cells, mainly erythrocytes, were removed by changing medium. The primary preadipocytes were differentiated into adipocytes using differentiation culture medium. Adipocytes and preadipocytes were confirmed by microscopy and oil red O staining.【Results】Rat adipocytes were cultured by modified ceiling culture method in vitro and identified by microscopy and oil red O staining. These adipocytes were highly homogenous, stable, numerous; Culture method of preadipocytes was improved and preadipocytes obtained by this method were able to proliferate and differentiate into adipocytes. Oil red O staining and morphological observation verified these cells to be preadipocytes. The preadipocytes were highly homogeneous, proliferative and could differentiate into adipocytes.【Conclusions】Adipocytes cultured by the modified ceiling culture method were highly homogenous, stable, numerous; and preadipocytes showed highly homogeneous appearance with active proliferation, and could differentiate into mature adipocytes,having the typical characteristics of preadipocytes. The modified culture methods of adipocytes and preadipocytes are simple, stable and easily conducted. PART TWO HIGH-FAT DIET-INDUCED OBESE RATS MODEL【Objective】To establish high-fat diet-induced obese rats model and observe the different weight increase patterns of high-fat diet and characteristics of biomarkers【Subjects and Methods】A group of 40 male Sprague-Dawley rats (SD rats) at the age of 28 days were used. We randomized 40 rats into standard chow group (n=10) and high-fat diet group (n=30). Body weight of rats in both groups was measured for 12weeks on a basis of 7 days interval. At end of the study, rats were killed and blood samples were drawn via femoral vein. Serum insulin, leptin, glucose, total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured. Epidedymal and perirenal fat pads were weighed.【Results】After initiating high-fat diet feeding, body weight growth in rats between control (fed with standard chow) and experiment (fed with high-fat diet) groups shown different growth patterns. Rats fed with high-fat diet grown quicker than the rats fed with normal diet. At end of the study, i.e. 12 weeks of experiment, body weight and body weight gain of rats fed with high-fat diet were 119.8% and 126.0% of the rats fed with normal diet, respectively. Rats fed with high-fat diet were categorized into three groups, i.e. diet-induced obesity (DIO), diet-induced resist (DR), and non-categorizable (NC) rats according to their body weight gain,12 rats were defined as DIO rats, 9 rats were defined as DIO rats, 9 rats were NC rats. From two to three weeks of experiment on, body weight growth of DIO, DR, and normal rats was separated, remaining in a descending order of DIO, DR, and normal until the end of the study. Individual growth and growth gain of rats in the three groups was completely separated, i.e. no any overlaps after eight weeks of experiment. No statistical significant differences were identified between rats fed with the two diets and among DIO, DR, and NC rats in serum insulin, TC, HDL, and LDL. However, significant higher (P<0.05) serum levels of leptin, glucose, and TG were found in rats fed with high-fat diet compared with rats fed with normal diet. In addition, significant higher leptin and glucose levels were observed in DIO rats than in DR rats as well. Fat pads were heavier in rats (any group of the DIO, DR, or NC groups) fed with high-fat diet than that in rats fed with normal diet. Furthermore, perirenal fat pads were heavier in DIO rats than in DR rats, but not for epidedymal fat pads.【Conclusions】High-fat diet induced rats to become obese. For a period of 12 weeks, high-fat diet resulted in 20% to 26% more weight gain compared with normal diet. The first 6-8 weeks was the main contributory period for obese development in rats. Different weight increase response patterns to high-fat diet were observed in the rats studied. After eight weeks, DIO and DR rats were correctly identified. Elevated serum levels of leptin, glucose, and TG were determined in rats fed with high-fat diet. DIO rats had high levels of leptin and glucose compared with DR rats. PART THREE MIGRATION AND DIFFERENTIATION OF MESENCHYMAL STEM CELLS IN VIVO TO ADIPOSE TISSUE IN RATS FED WITH HIGH-FAT DIET【Objective】To identify migration of mesenchymal stem cells (MSCs) to adipose tissue and their differentiation to adipocytes in vivo in rats fed with high-fat diet.【Subjects and Methods】A dose of 1×106 cells/100g green fluorescent protein-expressing MSCs (GFP-MSCs) was transplanted into homologous male SD rats at the age of 28 days. All rats were fed with 75% high-fat diet and 25% standard chow. After 7, 8, 9, 10, 11 weeks of transplantation, rats were killed and adipose tissues were excised, fat tissue slides were stained by DAPI and detected by laser confocal fluorescence microscopy. Immunohistochemistry and Western Blotting were used to identify GFP.【Results】Cells expressing both green fluorescence and blue fluorescence were observed under the laser confocal fluorescence microscopy in the rat fat tissue slides. The cells were defined as adipocyte based on the cell morphology and their tissue sources. The cells were scattered distribution. Comparing with other fat tissues, mesenteric adipose tissue contained more cells expressing green and blue fluorescence. GFP was detected by immunohistochemistry, Western Blotting method as well. 【Conclusions】MSCs can migrate to adipose tissue and differentiate to adipocytes in vivo in rats fed with high-fat diet.