| Chapter One Studies on the Expression and Secretion of Transforming Growth Factor-beta2 by Human Retina Pigment EpitheliumObjective:To measure the level of transforming growth factor-beta2 (TGF-beta2)expressed and secreted by human retina pigment epithelium (RPE)cell line D407 under the common culture condition.Methods:The human RPE cell line D407 was cultured in dulbecco's modified eagle's medium(DMEM)with 10%calf serum under the common condition(37℃,5%CO2).The supernatant and cells were collected at the exact time points(2,4,8,16,24 and 48h)after the medium changed.The location of TGF-beta2 protein was determined by immunocytochemistry.Levels of TGF-beta2 mRNA and protein in cytoplasm and concentration of TGF-beta2 protein in supematant were assayed by reverse transcription polymerase chain reaction(RT-PCR), Western-blot and enzyme linked immunosorbent assay(ELISA), resectively.The data was analyzed by means of analysis of variance (ANOVA).Results:TGF-beta2 protein was localized in cytoplasm of D407.The differences in levels of TGF-beta2 mRNA and protein among all time points were no statistics significance(P＞0.05).The content of TGF-beta2 protein in supernatant was upgraded at 4h after medium changed and reached to peak at 24h(586.9±28.5pg/mL).There was significant difference in contents of TGF-beta2 protein in supernatant among all time points.(F=86.593,P＜0.001). Conclusions:The TGF-beta2 protein of human RPE cell localized in cytoplasm.RPE cells could express TGF-beta2 steadily and secreted it.Chapter Two The Effect on Cell Proliferation of Carbachol and Atropine to Human Retina Pigment EpitheliumObjective:To establish the concentration ranges of carbachol and atropine refer to studying the function of human RPE cells by observing the changes in morphology and proliferation rate of D407.Methods:D407 cells were cultured in dulbecco's modified eagle's medium(DMEM)with 10%calf serum under the common condition (37℃,5%CO2)and seeded in 96-well plate with 5×103 cells per well.For synchronization,cells had been cultured in serum-free medium(SFM) after adherence for 24 hours,then divided into 3 groups:(1)Group A1～6: Cells were treated with carbachol(10-8mol/L～10-3mol/L);(2)Group B1～6:Cells were treated with atropine(10-8mol/L～10-3mol/L);(3)Group C:Cells were untreated and cultured with SFM conventionally as the control.The morphology of cells was observed by contrast phase microscope,and the proliferation rate was measured by methyl thiazolyl tetrazolium(MTT)chromatometry per 24h in 3 days.The data was analyzed by means of repeated measure analysis of variance.Results:Carbachol didn't show significant effect on the change of cell morphology,but could promote cell proliferation instead.The light absorption value(A)was higher follow the prolongation of action time. There was significant difference in value A among time points (F=3200.150;P＜0.001)and groups(F=3.324;P＜0.05).LSD test showed that there are significant differences between any two time points (P＜0.001),or between group A6 and any other groups(P＜0.05).No interaction was found between the concentration of carbachol and action time(F=0.307;P＞0.05).Cells became round and shrinkage with contour enhancement under the treatment with 10-3mol/L atropine.There was toxic granulation in the cytoplasm,accompanying with significant decrease of cell proliferation. Part of cells could return to normal appearance after 48h or 72h,but the abnormal cells also could be seen.There were no marked change of the morphology with the treatment of 10-8mol/L～10-4mol/L atropine.The light absorption value(A)was higher follow the prolongation of action time.There was significant difference in value A among time points (F=6999.917;P＜0.001)and groups(F=25.726;P＜0.05).LSD test showed that there are significant differences between any two time points (P＜0.001),or between group B6 and any other groups(P＜0.05).The interaction between the concentration of carbachol and action time could be seen(F=2.708;P＜0.01).Conclusions:Carbachol could promote the proliferation of RPE cells when its concentration reached to 10-3mol/L.10-3mol/L atropine could inhibit the proliferation of RPE cells obviously.Both carbachol and atropine could be used to investigate the function of RPE safely with the concentration of 10-8mol/L～10-4mol/L.Chapter Three TGF-beta2 Expression and Secretion in Human Retinal Pigment Epithelium Cells Regulated by CarbacholObjective:We studied the regulation of carbachol on the expression and secretion of transforming growth factor beta2(TGF-beta2)of human RPE cells by observing the time- and dose-effect relationship of the expression and secretion of TGF-beta2.Our aim is to elucidate cholinergic neurotransmitters could participate in the cascade transmission of myopic signals.Methods:D407 cells were cultured conventionally same as above and divided into two groups.One was incubated with carbachol(10-8～10-4mol/L)as experimental group,the other was untreated as the blank. The concentration of TGF-beta2 in the supernate,the level of TGF-beta2 mRNA and protein induced by carbachol were measured by ELISA, RT-PCR and Wetern blot at 2,4,8,16,24 and 48 hours,respectively.And the time- and dose-effect relationships were explored.The data was analyzed by means of repeated measure analysis of variance.Results:The level of TGF-beta2 mRNA and protein and the concentration of TGF-β2 in the supemate were higher follow the prolongation of action time and there were significant differences among time points(F=4455.148,2619.21,3280.75;P＜0.001).LSD test showed that there were significant differences between any two time points (P＜0.001).All index also up-regulated with the increase of the concentration of carbachol and there were significant differences among time points(F=1102.240,2918.62,448.45;P＜0.001).LSD test showed that there were no significant differences in the level of TGF-beta2 mRNA among A1,A2 and A3 group,the level of TGF-beta2 protein between A2 and A3 group,or A4 and A5 group,and the concentration of TGF-beta2 in the supernate among A3,A4 and A5 group(P＞0.05).The interaction between the concentration of carbachol and action time could be seen(F=249.609,186.02,92.96;P＜0.001).Conclusions:Carbachol could enhance the function of expressing and secreting TGF-beta2 of human RPE cells.The effect showed time and dose dependence.Besides,the tendency with time of this effect was influenced by the concentration of cabarchol.It is suggested that cholinergic neurotransmitters maybe participate in the cascade transmission of myopic signals by promoting the expression and secretion of those signals of RPE.Chapter four TGF-beta2 Expression and Secretion in Human Retinal Pigment Epithelium Cells Regulated by AtropineObjective:The present study was conducted to investigate the regulation of atropine to TGF-beta2 expression and secretion of RPE cells and try to find its probable mechanism and action site of delaying the development of myopia.Methods:D407 cells were cultured conventionally same as above and divided into three groups as follows:(1)experimental group(group A1～5):cells were pretreated with 10-4mol/L～10-8mol/L atropine for 30 minutes,respectively,then treated with 10-5mol/L carbachol;(2)positive control group(group B):cells were treated with 10-5mol/L carbachol;(3) blank control group(group C):cells were untreated.The concentration of TGF-beta2 in the supernate,the level of TGF-beta2 mRNA and protein induced by carbachol were measured by ELISA,RT-PCR and Wetern blot at 24 hours after treatment.The data was analyzed by means of analysis of variance(ANOVA).Results:There were significant differences in the level of TGF- beta2 mRNA,protein and the concentration of TGF-beta2 in the supernate among groups(F=1056.897,1320.170,475.657;P＜0.001). LSD test showed that there were no significant differences in the level of TGF-beta22 mRNA between A1 and C group,or A5 and B group;the level of TGF-beta2 protein between A1 and C group,or A3 and A4 group;and the concentration of TGF-beta2 in the supernate between A1 and C group, or A3 and A4 group(P＞0.05).Conclusions:Carbachol could promote the expression and secretion of TGF-beta2 of human RPE cells via M receptors and atropine could reverse it effectively.It is suggested that the antagonists of cholinergic M receptors probably block the development of myopia by this way.
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