The Research On Functions Of PI3K/Akt/mTOR Signal Pathway Inhibitor In The Retinal Pigmentepithelium Cells Proliferation Mechanism

Objective:Exploring whether the expression and significance of PI3K/Akt/mTOR signal pathway that are in human being proliferative vitreoretinopathy?PVR?,and detecting the expression of PI3K?Akt?mTOR?P70S6K though human being D407RPE cell line in vitro experiment,and making experimental treatment to RPE abnormal proliferation by applying the specific inhibition of mTOR agent Rapamycin?Rapamycin,RAPA,Rapa?,PI3K inhibitor LY294002,in order to exploring its dose?time and mechanism of action.Methods:1.MTT method of exploring cell inhibition ratioFrom logarithmic phace of RPE cells per 5×10⁴ hole in 96 cells in cell culture plate,37?,5%CO₂ Conditions cultivate.All cells are divided into six experimental group and control group,the concentration of RAPA;30ng/ml,90ng/ml,180ng/ml.LY294002concentration:15?mol/L,30?mol/L,45?mol/L respectively in the experimental group6,respectively,cultivate 6,12h,24h after the 490nm wavelength used to measure the value ofeachholeOD?absorbancevalue?.Atthesametime,zero-hole set?medium,MTT,DMSO?,the control hole?cell culture medium,MTT,DMSO?,eachset six-hole complex.Calculation of inhibitionrate=[?control-blank?-?drug delivery-the blank?]/?control-blank?×100%2.Flow cytometry exploring cell proliferation index?cell cycle and apoptosisEmpty control group:newborn calf serum RPMI-1640.The control group:No cell culture+RAPA group Cell culture+RAPA group:the concentration of RAPA:90ng/ml,add drugs in cell culture group:LY294002 concentration:30?mol/L respectiv-ely cultivate 6h,12h,24h,by flow cytometry detection of cell proliferation FAC Star Index,three cases in each group.Calculating the percentage of S+G2/M phase cells in the total number of occupied cells?G0/G1+S+G2/M?,that is the degree of cell proliferation.Statistical analysis,experimental data are shown by the standard deviation?x±S?the experiment repeated 3 times.To compare the difference among groups,using SPSS 13.0statistical analysis software,and paired t test.3.RAPA,LY294002,cell morphology observation before and after treatment:By inverted microscope observation of RPE cells were untreated group and 90ng/ml RAPA,LY294002 treatment group status 24h of cell growth and morphological changes.4.RT-PCR,western blot test PI3K?Akt?mTOR?P70S6K?4EBP-1 the mRNA and protein expressed in tissue and cells.Tissue sub-groups:control group?normal retinal group?:15cases that removed immediately eye ball.Ocular trauma eyes proliferation group:30 cases diagnosed diabetic retinalpathy combined with PVR1.30 cases ocular trauma combined with PVR2 above C degree at the PVR degree criteria cell sub-group:control group:without treatment cultured RPE cellsthe experimental group 1:RAPA 90ng/ml add 24 cultivate the RPE cellsthe experimental group 2:add LY294002 30?mol/lof RPEcultivate fine 24h cell.Results:1.Cell proliferation?MTT?test results:dCompared different concentrations of RAPA,LY294002 treatment of cells with untreated cells.OD values wwere significantly different?p<0.05?,the higher concentrations of RAPA,the lower cell OD values,the slower cell proliferation inhibited.It means RAPA,LY294002 significantly inhibited cell proliferation and with the increase of RAPA,LY294002 concentration,the inhibitory rate of cells increased,the same concentration of RAPA,LY294002 treatment of cells,24h's inhibition rate is bigger than12h,6h's inhibition rate.2.Flow cytometry test the effects of RAPA and LY294002 on cell cycle and apoptosis:Compared with the untreat group RAPA,LY294002 significantly inhibited the cell in G1 phase,and with RAPA,LY294002 role of time increased,stagnation in the number of G1 phase cells increased.It showed that RAPA,LY294002 can inhibit cell cycle progression,thereby inhibiting cell growth.On the other hand,RAPA,LY294002treatment in the synthesis phase that is significantly lower number of cells?p<0.05?,further indicates that RAPA,LY294002 on cell growth inhibition.3.RAPA,LY294002 cell morphology observation before and after treatment:RAPA,LY294002,after 24h treatment,compared with untreated cells,cells were significantly changed,cells become smaller and longer and there is floating cells,cell proliferation become slower.4.Test PI3K?Akt?mTOR?P70S6K?4EBP-1 the mRNA and protein expression by means of RT-PCR,western blottingRT-PCR,western blotting results showed PI3K?Akt?mTOR?P70S6K inproliferation membrane increased,4EBP-1 expression reduced,the difference have statistical significance.RAPA made mTOR?P70S6K expression reduced,4EBP-1 increased,?p<0.05?.To developing RPE cell,the difference have statistical significance,no obviviously effect on PI3K?Akt expression.LY294002 made PI3K?Akt?mTOR expression reduce,4EBP-1 expression decrease.the difference have statistical significance.Conclusion:1.PI3K/Akt/mTOR signaling pathway expressed in retinal tissue,,stronger expre-ssion in proliferation membrane.2.PI3K/Akt/mTOR expressed in signaling pathway in RPE cell growth.3.RAPA blocked specific signaling patway,reducee the expression of mTOR?P70S6K and inhibition of 4EBP-1 phosphorylation,cell cycle arrest at G1 phase.4.PI3K specific inhibitor LY294002 can block specific phosphorylation procession ofPI3K...