The Effects Of Leptin, Insulin And IGF-2 On The Expression Of PKB And P⁷⁰S6K Protein In The Trophoblastic Cells And The Signaling Pathway

ObjectiveSince the birth weight is closely related to adult diseases,Present investigations on the birth weight indicated that the birth weight was regulated by nutrition,the structure and function of placenta,growth factors and hormones,among growth factors and hormones the most representative factors were leptin,insulin and insulin-like growth factor -2(IGF-2),these three factors have certain regulative functions in the birth weight where is the mechanism for the kind of regulatory functions? PI3K/PKB/mTOR signaling pathway can regulate the growth and differentiation of the trophoblastic cells by regulating the phosphorylation of p⁷⁰S6K.This investigation discussed whether the effects of leptin,insulin and IGF-2 on the birth weight have correlations to the PI3K/PKB/mTOR pathway by studying the expression of PKB and p⁷⁰S6K protein in the trophoblastic cells and the regulatory functions of specific inhibitors in the PI3K/PKB/mTOR signaling pathway,namely LY294002 and rapamycin.Material and MethodsThe villi were collected from the normal pregnant women had been pregnant for 6 to 8 weeks were digested with 0.25%trypsin at 4℃for about 40 mins.25 IU/ml DNase I was added for 10-15 mins after the centrifugation.Then it was sieved by a strainer mesh of 100 screen mesh and subjected to a density gradient sedimentation with 2.5% and 1.25%BSA for 45 mins.The trophoblastic cells in the underlayer were collected after the centrifugation and they were inoculated in a culture flask that had been overlaid typeâ… collagen after another centrifugation.The culture flask was incubated in an incubator containing 5%CO₂ at 37℃and the relative humidity was 99%.The culture solution was changed once for every two or three days according to the conditions of the cells.The trophoblastic cells during the first trimester in good conditions were selected and DMEM nutrient solution containing 10%fetal bovine serum was changed after the cells were starved for 24 hours.The cells were subjected to treatments with 10nmol/L leptin;6nmol/L insulin and 25nmol/L IGF-2 for 24 hours, respectively.20μmol/L LY294002 and 100nmol/L rapamycin were added to the other group for 24 hours simultaneously.The cells(5-6×10⁶) were collected after 24 hours for use.According to the procedures for Kaiji kit of total protein extraction.The protein concentration was determined by Coomassie brilliant blue method.Western blot was used to detect the expression of PKB and p⁷⁰S6K in the trophoblastic cells after dealt with leptin,insulin and IGF-2;the effects of leptin,insulin and IGF-2 on the expression of PKB and p⁷⁰S6K were re-examined after the addition of rapamycin and LY294002. Gene Tools from Syngene gel image analysis system was used for the systematic result analysis,the data were represented by(?)±s,and SPSS13.0 statistical software was used for ANOVA,and the difference was statistically significant(P＜0.05). Results1.Compared with control group,the expression of PKB and p⁷⁰S6K in test group were significantly raised(dealt with leptin,insulin and IGF-2)(P＜0.05).2.The expression of PKB and p⁷⁰S6K in the trophoblastic cells after added LY294002 or rapamycin were significantly decreased in test group(dealt with leptin, insulin and IGF-2)(P＜0.05).Conclusion1.Leptin,insulin and IGF-2 can promote the expression of PKB and p⁷⁰S6K protein in the trophoblastic cells and the promoting effects were significantly inhibited after the specific inhibitor in the PI3K/PKB/mTOR signaling pathway,namely rapamycin and LY294002,were added.2.The effects of leptin,insulin and IGF-2 on the birth weight may be realized partly by the PI3K/PKB/mTOR signaling pathway.