The Effects Of IGF-1 On Proliferation,Apoptosis And Apoptosis Modulin Bax,Bcl-2 Expression Of MC3T3-E1

Object:1. To investigate the effect of insulin-like growth factor-1(IGF-1) at different interventional concentration on proliferation of MC3T3-E1 under serum deprivation.2. To demonstrate the effects of insulin-like growth factor-1(IGF-1) at different interventional concentration on apoptosis and apoptosis gene regulated proteins Bax, Bcl-2 expression of MC3T3-E1 under serum deprivation.Method:1. Serial subcultivation of Murine preosteoblastic line MC3T3-E1.2. Murine preosteoblastic line MC3T3-E1 cells were cultured in experimental designed groups according to supplements in DMEM and treated with different dosages of IGF-1(1,10,30,50ng/ml) during 24 hours. The dynamic growing MC3T3-E1 cells of all groups were observed with the microscopy:When treatment started, osteoblasts were incubated in the following six conditions: Normal serum control group: used DMEM basal medium containing 10% fetal calf serum; Serum deprivation control group: used DMEM basal medium containing 0% fetal calf serum; Four designed-dosage IGF-1 groups: used DMEM basal medium containing 0% fetal calf serum. When observing MC3T3-E1 cells conjugation to reach 40-50% under the inverted phase contrast microscope, MC3T3-E1 cells of IGF-1 groups were intervened by adding corresponding concentration.3. The growing MC3T3-E1 cells were measured by MTT colorimotric assay. The apoptosis rate and cell cycle were analyzed by flow cytometry. Bax,Bcl-2 expression were stained by immunocytochemistry and determined by computer image analysis system.4. Statistical analysis:All experimental date are presented as x±s .Statistical analysis was performed with Windows SPSS11.5. The differences between the groups were determined by One-way ANOVE.Different sample means were compared by applying LSD-t test. Moreover the correlation of the expression density ratio of Bax/Bcl-2 and apoptosis of MC3T3-E1 was analyzed by adopting Pearson's ranking test. Differences were considered significant at a value of P<0.05.Results:1. Compared with normal serum concentration group ,the proliferation of MC3T3-E1 cells decreased in serum deprivation group(P<0.01),the percentages of S+G2-M phase cell did also(P<0.01),yet the percentage of apoptotic MC3T3-E1 cells increased(P<0.01);2. Compared with serum deprivation group, 10-50ng/mlIGF-1 added declined the inhibition of the proliferation of MC3T3-E1 cell(sP<0.01),with the growth rate of MC3T3-E1 cells gradually increased according to a dose-dependent relation. 30-50ng/ml dosage could decrease the percentages of G0-G1 phase cells(P<0.01)and the percentages of S phase cells significantly increased at 50ng/ml concentration group(P<0.01).3. Compared with serum deprivation group, while increased Bcl-2 expression of MC3T3-E1 cells(P<0.05)and decreased Bax expression(P<0.01)with reduced apoptosis of MC3T3-E1 cells(P<0.01)after IGF-1 treatment; Four designed-dosage IGF-1 groups compared , it could been seen that there was a obviously increased Bcl-2 expression(P<0.01)at 50ng/ml IGF-1 group when Bax expression(P<0.05)and the apoptosis(P<0.05) gradually decrease with designed-dosage increased. There was a remarkable positive relationship between the expression density ratio of Bax/Bcl-2 and the apoptosis rate.Conclusions:1. The proliferation inhibited and the apoptosis promoted when MC3T3-E1 cells were been cultured under serum deprivation condition.2. A certain designated dosage IGF-1 could prevent inhibition of MC3T3-E1 cells under serum deprivation and stimulate proliferation of MC3T3-E1 cells.3. A certain designated dosage IGF-1 could inhibit the apoptosis rate of MC3T3-E1 cells induced by serum deprived. Participating in the apoptotic mechanisms of MC3T3-E1 cells, both Bax and Bcl-2 can been regulated by IGF-1. There exited a dose-dependent effect.4. Both promoted the proliferation and inhibited the apoptosis of MC3T3-E1 cell after IGF-1 treatment .The double-acting of IGF-1 to osteoblast is maybe one of the mechanisms for promoting bone formation and prevention and curing osteoporosis.