| Objective: To observe the effects of Janus kinase/signal transducer and activator of transcription pathway (JAK/STAT pathway) inhibitor AG490 on the proliferation of human fat cells in vitro,and to understand the role of AG490 to human fat cells or not. To applicate different concentrations of AG490 on cells in the higher physiological leptin concentration(100ng/ml) and intervene cells adipocyte lipid metabolism, and explore the formation mechanism of leptin resistance caused by JAK/STAT pathway.Methods: (1) Preadipocytes were isolated from human abdominal adipose tissue by collagenase digestion successfully. Their proliferation and differentiation process were observed in vitro. to be induced to differentiate into fat cells after cells were fused in bottles,cells were identified by their dynamic morphological changes using microscope and Oil Red O staining.divined adipocytes into some groups by random.(2) The proliferation process of adipocyte was interfered by four different concentrations, AG490(5,10,15,20umol/L). The effects of AG490 were detected by the methods of MTT at the time of 1d, 2d, 3d,4d. (3) The metabolic process of adipocyte cells in the higher physiological leptin concentration was interfered by four different concentrations, AG490(5,10,15,20umol/L). The effects of AG490 were detected by the measure the concentration of free fatty acid and glycerol in nutrient medium with optical densities at the time of 24h, 48h, 72h. (4) Analyze the data and observe the effects of AG490 on the proliferation of fat cells and metabolism of human adipocyte in the super-physiological leptin concentration.Results: (1) We successfully separated and primary cultured the human preadipocyte, and observed the proliferation and differentiation process of preadipocyte. The cells grew rapidly from the 4th to the 10th day. Lipid droplets were detected on the 4th dAy of differentiAtion. The cytoplasm were filled with more and more lipid droplets, And most preadipoctes differentiated into adipocytes on the 21st day;the passage time of matured fat cells was for 3-4 days.(2) The result measured by MTT showed AG490 in different concentrations(5,10,15,20umol/L)at the time of 1d,2d,3d,4d cannot stimulate or inhibit the proliferation of adipocye. (3)â‘ At the time of 24 hour , the concentration of free fatty acid in diffent AG490 concentrations group increased by 33.6%,35.6%,37.1%,37.5% about respective contrast to the control group;At the time of 48 hour , the concentration of free fatty acid in diffent AG490 concentrations group increased by 28.0%,33.9%,54.6%,54.4% about respective contrast to the control group;At the time of 72 hour , the concentration of free fatty acid in diffent AG490 concentrations group increased by 45.8%,63.0%,98.7%,100.1% about respective contrast to the control group(P<0.05).â‘¡At the time of 24 hour , the concentration of glycerol in diffent AG490 concentrations group increased by 46.9%,56.4%,79.9%,80.6% about respective contrast to the control group;At the time of 48 hour , the concentration of glycerol in diffent AG490 concentrations group increased by 18.7%,23.1%,44.2%,43.7% about respective contrast to the control group;At the time of 72 hour , the concentration of glycerol in diffent AG490 concentrations group increased by 15.1%,23.8%,28.5%,31.4% about respective contrast to the control group(P<0.05).â‘¢No significant diffent between the concentration of AG490(15umol/L)'s group and AG490(20umol/L)'s group(P>0.05).Conclusion: (1) We separated and primary cultured the human preadipocytes and induced to differentiate human fat cells successfully, And provided a model for studing the effects of hormone and drugs. (2)AG490 in different concentrations at the time of 1d,2d,3d,4d cannot stimulate or inhibit the proliferation of human fat cells;so we speculate there have no direct relationship between AG490 and Fat Cells' metabolism.(3)AG490 can interfere with fat cells with leptin on lipid metabolism and also have time-dependent and dose-dependent manners. |
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