Effect Of Tumor Necrosis Factor-α On Apoptosis Of Rat Adipocyte

Healthy, disease-free, 20-day-aged, SD-two-grade rats developing normally were selected as experimental material in this paper. Adipose tissue from perirenal, epididymal and inguinal was sampled in sterile condition. Preadipocyte was acquired after adipose tissue digested with collagenase Ⅰ. Preadipocyte inoculated at the concentration of 5(104 was cultured for 8-10 days in M199 medium (plus 10% fetal bovine serum, 37℃, 5%CO2). Trypan blue staining, HE staining, fluorescent staining, transmission electron microscopy (TEM), agarose gel electrophoresis, flow cytometry methods were adopted to study the influence of TNF-α on rat adipocyte apoptosis and the influence of different concentration of vitamin C and insulin on rat adipocyte apoptosis. The objective of this study is to supply basis for clarifying the metabolism of adipocyte apoptosis induced by TNF-α.The effects of TNF-α of different concentration on rat adipocyte apoptosis were detected, morphologic changes of rat adipocyte apoptosis induced by TNF-α correlated with the concentration of TNF-α linearly——within a determinate range higher concentration of TNF-α could induce more obvious apoptosis. The morphologic changes of rat adipocyte treated by 5 ng/mL TNF-α were clear, but the DNA electrophoresis didn't show the DNA ladders. This result indicated that the morphologic changes occurred more early than the biochemical changes. TNF-α induced the rat adipocyte apoptosis in a dose-dependent manner within a determinate range. Compared with the control group, the apoptotic rate of the group treated respectively by 5ng/mL, 10ng/mL, 15ng/mL, 20ng/mL TNF-α was seriously distinct (P<0.01), but that of the group treated respectively by 10ng/mL, 15ng/mL, 20 ng/mL was not distinct (P>0.05). Therefore, the optimum concentration of TNF-α inducing apoptosis was 10ng/mL.Rat adipocytes were treated respectively for 0,12,24,36,48hours with 10ng/mL TNF-α to oberseve the affects of different treatment times on rat adipocyte apoptosis. Flow cytometry was used to determine adipocyte apoptosis rate. The results show that apparent morphologic changes appeared with treatment time increasing, and terminal apoptosis dominated after culture for 48 hours. The stady also indicts that adipocyte apoptosis rate with treatment time increasing and appeared a increasing trendency. The difference between control and treatment groups was distinct (P<0.01). However, difference of apoptosis rate between 36 and 48 hours was not distinct (P>0.05), therefore, 36 hours maybe optimum time to induce adipocyte apoptosis by TNF-α.Effects of different Vc concentration on adipocyte apoptosis induced by TNF-α.The results show that high concentration of Vc (100μmol) lead to adipocyte apoptosis, but apoptosis was not detected by low concentration of Vc(5,10μmol). 5,10μmol Vc can inhibit adipocyte apoptosis incuced by TNF-α which has significantly lower apoptosis rate than that from treatment induced by only TNF-α.However high concentration of Vc (100μmol) can improve adipocyte apoptosis induced by TNF-α,and significanty different from treatment induced by only TNF-α.Influence of different concentration of insulin on adipocyte apoptosis induced by TNF-α was studied and the results show that low concentration of (0.5nmol/L) lead to adipocyte apoptosis, but apoptosis was not detected by high concentration of can insulin. Adipocyte apoptosis was improved by induction with 0.5nmol/L insulin and 10ng/mL TNF-α, which dramatically higher than treatment induced by TNF-α only (P<0.01). Adipocyte apoptosis was inhibited by 5nmol/L, 50nmol/L insulin and 10ng/mL TNF-α, which significantly lower than treatment induced by TNF-α only (P<0.01).