Regulation Of Rat Preadipocytes' Differentiation By Vimentin Intermediate Filament

(The College of Animal Science and Veterinary Medicine, Northwest Science and Technology University of Agriculture and Forestry , Yangling, Shaanxi, P.R.China.712100)Experiments of generating adipocytes from stromal-vascular (SV)cells of the fat tissue of SD rats had been successfully made in advance in order to investigate the methods of adopocyte culture and cellular biological characterization. Then the differences of types and distributions of the intermediate filaments (IF) between preadipocytes from adipose tissue and mature adipocytes from cell culture were studied by using the following techniques:selective extraction associtated with whole mount electronmicroscope, immunohistocytochemistry, in-situ-hybridization. immunofluorescent cytochemistry, SDS-polyacylamide gel electrophoresis and Western blotting. Thus, the effect of microinjection of anti-vimentin monoclone antibody into preadipocytes on the differentiation of preadipocne were oberserved, to elucidate the mechanisms on the regulation of IF associated with preadipocytes differentiation and provide a new and effective stratege for the regulation of fat overdcposition. This study mainly contains the following works: 1 Biological characteristics of differentiation of the rat presdipocytes in primary culture.Objective :To establish optimal conditions for the primary culture of rat adipocytes , serving as a model for studying human or other animal adipocyte development and metabolism in vitro , and elucidate the role of insulin through the preadipocyte-adipocyte transition, as well as the charaterization of morphology and biology of cultured preadipocytes. Methods:There are two approaches to culture adipocytes:tissue culture method and digestion enzymatically culture method. Adipose tissues were from perirenal ,epididymal and inguinal adipose depots of male 20d-old SD rats .Digestion culture method use Preadipocytes derived from stromal-vascular cells isolated from adipose depots- by collagenase I digestion , seeded at a density of5xl04 cells/cm2 in Ml99 medium plus 10%fetal bovine serum (FBS) and insulin (50 nM) and cultured at 37?under a humidified atmosphere of 95% air :5% CO2 for 10 days. Adipocyte differentiation was assessed by Changes in the morphology and number of adipocytes , stained by oil O red, and the biochemical components (totle lipid contenK the activity of Glycerol 3-phosphate dehydrogenase (GPDH) and triacylglycerol (TG)) measured by 7170A biochemistry analyzer , as an index of adipocyte . Inaddition, Adipose differentiation was induced by exposure to 0 nM - 50nM and 100 nM insulin, detected by MTT assay.Adipocytes (days 6-7)were passaged at the seed density of 5X 104cells/cm2. Results:(l) The outgrowth could be seen around the fat tissue after 1-3 weeks and converted to adipocytes after 1 month .The success rate of explant outgrowth was 12%;whereas about 10 days were required from the preadipocytes to adopocytes using the digestion method . The population double time and the attachment rate were 59.75h ,39.1% respectively.(2) The changes of differentiating preadipocytes in both cellular morphological and physiological phenotypes occurred .A large number of preadipocytes develop with no lipid accretion(days 0-2),and the values of the totle lipid content , TG and the activity of GPDH were zero . At days 3,the lipid droplet began to appearance . Up to lOdays , the totle lipid content and TG increased ,with the significant difference (PO.01) compared that of day 3, and the activity of GPDH of adipocytes (318 IU/mg)was 4-fold more abundant than that of preadipocyte(day 5). Differentiated fat cells exhibited an increase and the percentage of plurilocular adipocytes reach to 86.8% ?.3% . (3) A significant increase in proliferation rate was observed with culture time (PO.01) under the control of 50nM or lOOnM insulin, comparing to OnM insulin. However, there was no marked differences in the proliferation rate between 50nM and lOOnM insulin treatment.(4) while adipocytes(day 6-7) after passage rapidly lost their capacity to...