The Research Of Cell Apoptotic Signal Transduction On Cultured Human Retinal Pigment Epithelium Induced By Verapamil

IntroductionIn physiological circumstances, the retinal pigment epithelium (RPE) is a mosaic of polygonal cells interposed between the choroid and the neural retina and serves as the outer blood - retinal barrier regulating retinal homeostasis and visual function. The apical side of RPE cells closely associates with the outer segments of cones and rods, whereas the base of the cell attaches to Bruch's membrane. Normally RPE cells form aquiescent monolayer, but they retain the ability to divide and do so when placed in culture or when participating in wound repair. RPE cells proliferate readily in vivo in response to a variety of stimuli such as photocoagulation and/or retinal cryotherapy. In mild injuries, involving only the RPE and photoreceptor cells, proliferating RPE cells quickly reestablish a continuous monolayer. However, after severe injury that may be associated with ocular trauma or retinal detachment, RPE cells can be detached and consequently found in the vitreous. Once in this new environment, RPE cells have been shown to dedifferentiate and exhibit a pseudometaplastic transformation into fibroblast - like, spindle - shaped cells, which become actively dividing and migratory . These processes are considered to be key events in the onset of prolifer-ative vitreoretinopathy ( PVR). Normally RPE cells form a quiescent monolayer , but they retain the ability to divide and do so when placed in culture and polarity may be partially lost. Thus, dedifferentiated proliferative RPE cell cultures have been used to study the very early phases of PVR, both in vitro and in vivo.PVR is an excess proliferation which ocular tissue is in response to repair intrauma. Immigration and proliferation of RPE can promote PVR and RPE cells are the most important cells in forning proliferation membrane of PVR. The experiments including animal model and clinic inspect found that the nonprolifera-tion of RPE cells of PVR may be lost and differentiated into fibroblast - like and fibroblast - like cells. So RPE cells play important roles in development of PVR.The therapy of PVR in clinic is viteoretinal operation. Although the technique of viteoretinal operation promoted gradually, the recurrance rate is not improved. It is necessary to apply drugs or viteoretinal operation combined with drugs to inhibit the formation and development of PVR in the earlier peroid. RPE is the most important cell in developing PVR, the key point of blocking is controlling the proliferation of RPE. Recently, the hot research spot in PVR is controlling the proliferation of RPE by apoptosis. Applying the induced - apop-tosis agents make the migratory and proliferative RPE apoptosis and blocked fundamentally the formation and further development of PVR membrane. Daunomycin, hydrogen, peroxide, indomethacin and ratinoic acid can induce cultured RPE in vitro, apoptosis by different unknown molecular mechanism, but they are restrictly applied in clinic because of thire side effects. Verapamil is a kind of reliable calcium channel bloking agent and was applied extensivly in clinic, it has less side effects. We Previously applied calcium channel blocker -verapamil to induce cultrured human RPE in vitio apoptosis successly, but the mechanism of apoptosis and which apoptotic signals have involved in is unknown.Apoptosis indicats under the physiological conditions, organism cytogenic a series of changes of morphous and biochemistry and cause cytocidal course through many different signal transduction after post - stimulus, namely pro -grammed cell death ( PCD). But under the different circumstances and stimulant , apoptotic pathway in different species cells is distinct, so the apoptotic signal transduction pathway has versatility and make the apoptotic regulation mec -nanism more complex. With the investigation of apoptotic sinal transduction, we realised apoptotic sinal transduction is intercross of different pathway and interactive of different signals, it organize reticular signal network. Different genes,cell factors and some proteases participate in apoptosis. Calcium serve as actuating signal in many cells apoptosis. Bel - 2 family regulate the development of apoptosis in signal transduct pathway and regulate intracellule calcium through some mechanism. Caspase - 3 of caspase family is the executor of apoptosis and is regulated by Bel - 2. The activation of caspase family can hydrolysis Bel - 2 proteins and induce apoptosis. The activation of caspase - 8 may induce apoptosis through ectopathway.We previously applied different concentration Ver on cultured human RPE cells and found Ver can inhibited proliferation of RPE. 80mg/L Ver can induced RPE cells apoptosis, but how did calcium change, wether the change of calcium can actuate Bel -2? Wether caspase -3 and caspase -8 involved in apoptosis? How did Cell protective factor - bFGF change is unknown. Under this foundation , we make an initial explore of the apoptotic singnal transduction regulative mechanisms on this apoptotic model.Material and Methods— ^Tissue source and preparationUnder sterile condition, we obtain the donator after the cornea trans - plantation from the department of ophthalmology in the first affiliated hospital of China Medical University. Average ages were between 24 and 38 years.H^The primary culture human RPEUnder sterile condition, using enzyme digestive method to isolate and complete plantation of human RPE by 0. 25% trypsin to 24 - pore culture plate. The cells were transferred by 0.25% trypsin after they became 80% -90% confluent monolayer.Experiment one: Verapamil induced apoptosis in cultured human retinal pigment epithelium cells and changes of intracellular [ Ca + ] i.Planting the third period human RPE on 3. 5cm2culture dish, applying 80mg/L verapamil on cultured RPE for 12hA24h and 48h, control group was established simultaneously. Fluo - 3/AM load technique and MetaFluo4. 5/ cool-snapfx/IX70 intracellular Ca2 + fluorescence imaging system were used to detect. c .intracellular Ca2 + fluorescence of RPE cells after cocultured with 80mg/L verapamil between 10s and 10 min in each group. Intracellular calcium saturation of each group was calculated to observed the changes of intracellular [ Ca2 + ] i of RPE in apoptotic course.Experiment two: the expression of bcl - 2 and bFGF on apoptosis of cultured human retinal pigment epithelium cells induced by verapamil.Applying 80mg/L verapamil on cultured RPE for 12h^24h and 48h to induce RPE apoptosis, control group was established simultaneously. RT - poly-merase chain reaction( RT - PCR) was used to detect the mRNA expression of bcl - 2 and bFGF on apoptosis of cultured human RPE. The expression of bcl -2 protein was observed on RPE apoptosis by immunocytochemical staining(SP).Experiment three: the expression of caspase - 8 and caspase - 3 on apoptosis of cultured human retinal pigment epithelium cells induced by verapamil.Applying 80mg/L verapamil on cultured RPE for 12h^24h and 48h to induce RPE apoptosis, control group was established simultaneously. RT - poly-merase chain reaction (RT - PCR) was used to detect the mRNA expression of caspase - 3 and caspase - 8 on apoptosis of cultured human RPE. The protein expression of caspase - 3 and caspase - 8 was observed on RPE apoptosis by western blot.Automatic running gel imaging system was used to scan the straps of PCR and wstern - blot, optical density( OD) of each strap was read by FluorChen V. 2. 0 system. Cell image analysis system was used to analyse gray scale of bcl -2 stained by SP.Statistical Analysis: The date in this paper was represent in x ± s Analysis of variance was adopted to conduct statistics test. Statistics difference is P <0.05. Statistics was conducted by SPSS11.5 software.ResultsExperiment one: Primary cultured human RPE morphology showed round, cell body lucence and contain many black pigment granules. CeU morphology became flat and irregularity polygon, hyalin round cell nucleus and binucleatewere observed when adherencing. Intracytoplasmic black pigment granules gradually decreased after passage. RPE cell membrane shrinked after cocultured with verapamil, vacuolus were found in intracytoplasm. The fluorenscence in resting RPE cells was strong and distributed throughout the cells. Trie nucleus appeared more fluorescent than the cytoplasm. There was no change of intracel-lular Ca + fluorescence in normal RPE cells after cocultured with 80mg/L verapamil. Intracellular [ Ca2+ ] i ( nmol/L) of each group were 197. 25 ± 29. 03 , 176.09 ±25. 88,156.45 ±20.60 and 135. 28 ±21.18(P<0.01). Calcium fluorescence attenuated after cocultured with verapamil.Experiment two: Higher expression of bcl - 2 mRNA was observed in normal RPE and decreased after coculturing with verapamil for 12h, but there was no statistic difference compared with contrast group (P >0.05). The expression of bcl - 2 mRNA obviously droped with the prolong action time of verapamil. Bcl- 2 was detected in normal cultured human RPE cells, no significant change was found at 12h after cocultured with verapamil. But at 24h and 48h, a significantly decreasing of bcl - 2 expression was observed ( P < 0. 01). Bcl - 2/(3 -actin of each group were 1.14 ± 0. 16, 1. 03 ± 0. 17, 0. 66 ± 0. 04 and 0. 58 ± 0. 02. The expression of bcl -2 protein was paralleled to the expression of bcl -2 mRNA. The expression of bFGF mRNA increased gradually after cocultured with verapamil. bFGF/(3 - actin of each group were 1. 14 ± 0. 16,1. 03 ±0.17, 0. 66 ± 0. 04 and 0. 58 ±0 02. There was statistic difference( P <0.05 ).Experiment three: A lot expressions of caspase - 3 mRNA and protein were observed in normal RPE cells and increased after cocultured with verapamil. Caspase - 3/p - actin of each group were 0.58 ± 0.08,0. 89 ± 0. 12,1. 22 ± 0. 14 and 1. 18 ±0. 09. There was significant statistic difference in each group(P> 0. 01). Under our experimental conditions, we didn't find expressions of caspase - 8 mRNA and protein.Conclusions1. In the course of RPE apoptosis induced by verapamil, intracellular [ Ca2 + ] i decreased. Homeostasis disturbance of intracellular Ca + may be actu-ating signal of RPE apoptosis.2. In the course of RPE apoptosis induced by verapamil, both Bel -2 and bFGF are closely related to apoptosis involved in signal transduction pathway .3. In the course of RPE apoptosis induced by verapamil, caspase - 3 of caspase family play provital role in apoptotic signal transduction pathway, we didnt find expressions of caspase - 8 mRNA and protein. Verapamil induced RPE apoptosis by intracelluar pathway.