| Objective: Recent studies have suggested that obesity is a state of chronic low-grade inflammation, Adipose tissue could secrete kinds of inflammatory mediators. The aim for our research is to explore the effect of human tumor necrosis factor-α(TNF-α) on the proliferation of human preadipocytes and differentiation,apoptosis of adipocytes. This research is to investigate the relationship between obesity and TNF-α.Methods: Human preadipocytes have been obtained from abdominal greater omentum of surgery suffers and enzyme digestion. The proliferation of preadipocytes was detected with CCK-8 after treatment with TNF-αat the concentrations of 0,0.1,1,10,100ng/ml for 12,24,48 hours. The adipocytes differentiation by TNF-αat different concentrations on the 10th day has been observerd and the level of differentiation has been done with oil red o. Different concentrations of TNF-α.(0 ng/ml,1 ng/ml,10 ng/ml,100 ng/ml)have been used to detect the effect on fat cells after 36 hours treated.In the same time,the flow cytometry have been done to detect apoptosis in the situation.A concentration of 10ng/ml TNF-αhas been used to see the acts on preadipocytes for 0 hours, 12 hours ,24 hours ,36 hours and 48 hours. The flow cytometry method has been employed to detect apoptosis situation.Semi-quantitative western blot has been used for determination of the quantity about NF-KB in the fllowing groups, A:control group, B: TNF-αgroup ,C: TNF-α+ block group .GAPDH was used as an internal reference(n=4).Results:(1) TNF-αcan significantly promoted the proliferation of preadipocytes (P<0.05), especially the group treated with the concentration of 10ng/ml TNF-αfor 24 hours. (2)The differentiation of human adipocytes was reduced by TNF-α(P<0.05), significantly at 100ng/ml. (3) Flow cytometry results showed that TNF-αcould induced the cyto apoptosis in the best fit concentration of 10 ng/ml,and it could be observed the apoptosis of cells from the time after treated from the 12 h,apoptosis ration was increased from 12 to 48 h,after treated with TNF-α10ng/ml.The ration reached the top for the cell apoptosis in 36 h after the 10ng/ml TNF-αtreated.There was no statistical significance between 36 hs and 48 hs after 10ng/ml TNF-αused in the cell apoptosis ration.(4)The Western blot results of NF-KB in the three groups differed significantly(p<0.01).Group B(50.34±1.62,P<0.001) was higher than group C(19.78±3.67, P<0.001),group B was higher than group A(33.17±1.32, P<0.001),and group A was higher than group C(p<0.01).Conclusions: TNF-αcould effectively promote the proliferation of primary cultured preadipocytes and inhibit the differentiation of the adipocytes. TNF-αalso could induce the apoptosis of fat cells in a dose dependent way. TNF-αstill can active the IKK-IKB-NF-KB pathway which is a key pathway for the insulin resistance.It could be seen that the TNF-αwas playing an important role in the mechanism of obesity and insulin resistance. |
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