Effects Of Leptin On The Proliferation And Differentiation Of Human Preadipocyte

Objective: To observe the effects of leptin on the proliferation and differentiation of human preadipocyte in vitro, and approach the possible mechanism of leptin in the generation of obesity.Methods: (1) Preadipocytes were isolated from human abdominal adipose tissue by collagenase digestion. Their proliferation and differentiation process were observed in vitro. Cells were identified by their dynamic morphological changes, growth curve, and Oil Red O staining. (2) The proliferation process of preadipocyte was interfered by three different concentrations, leptin(10ng/ml, 100ng/ml, 1000ng/ml). The effects of leptin were detected by the methods of MTT and cell counting at the time of 24h, 48h, 72h. (3) The differentiation process of preadipocyte was interfered by three different concentrations, leptin(10ng/ml, 100ng/ml, 1000ng/ml). The effects of leptin were detected by the method of extracting stained intracytoplasmic lipid with Oil Red O at the time of 4d, 8d, 16d. The expression of PPARγ2 and C/EBPαmRNA was detected by RT-PCR at the time of 8d. (4) Analyze the data and observe the effects of leptin on the proliferation and differentiation of human preadipocyte.Results: (1) We successfully separated and primary cultured the human preadipocyte, and observed the proliferation and differentiation process of preadipocyte. They looked like fibroblasts. 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. (2) The result measured by MTT displaied leptin in higher concentration can stimulate the proliferation of preadipocye, and the effects increased by 7%, 12.8% and 5.7% respectively contrasting to the control group at the time of 24h, 48h, 72h. There are not obvious effects on the proliferation in the groups of lower and common concentration. It was also be found that leptin in higher concentration can stimulate the proliferation of preadipocye by cell counting at 48h. (3) The result measured by Oil Red O staining indicated that leptin in higher concentration can stimulate the differentiation of preadipocye at the time of 4d and 8d, and the effects increased by 13% and 23.2% respectively contrasting to the control group, but the effects were not obvious at 16d. And there are not obvious effects on the differentiation in the groups of lower and common concentration. It was also be found that leptin in higher concentration can stimulate the level of PPARγ2 and C/EBPαmRNA at 8d.Conclusion: (1) We separated and primary cultured the human preadipocytes successfully, and provided a model for studing the effects of hormone and drugs. Leptin in higher concentration can stimulate the proliferation and differentiation of human preadipocye in vitro. It maybe indicate that in physiological condition leptin effects the balance of body fat through acting on feeding center and the metabolism of lipid, and have little effects on the generation of adipocyte. Leptin maybe regulate the generation of obesity through acting on the proliferation and differentiation of preadipocyte at the state of leptin resistance and high leptin concentration in serum.