| PurposeProliferative vitreoretinopathy(PVR) is the common complication and the main cause of failure of rhegmatogenous retinal detachment (RRD) operation. Although the technique of vitreoretinal operation promoted gradually, the recurrence rate is not improved. And Vitrecto-my itself is a main risk factor in inducing the formation of PVR. Therefore, how to use medicines to inhibit the PVR& formation and development in the earlier period is an important problem need to solve. Retinal pigment epithelium ( RPE) is the most important cell in developing PVR. The key point of blocking PVR is inhibiting the proliferation of RPE. Recently,the hot research spot in PVR is controlling the proliferation of RPE by apoptosis . That is Adding the induced ?apoptosis agents to make the RPEs with migration and proliferation apoptosis. so that the formation and further development of PVR membrane are blocked fundamentally. At present, the drug of inducing RPE apoptosis is mainly Antimetabolite, which has great side effect and is not easy to accepted by patients. Verapamil is a kind of reliable calcium channel blocking agent, which has small side effect. And it is cheap, convenient and easy to be obtained. It is known that verapamil has double adjustment effect to the apoptosis of other tissue cells. Meanwhile, the function of the Ca + in apoptosis is not very clear. As we know, till now, there is no report about the induced function of verapamil to theRPE apoptosis and how it works in domestic and oversea areas. Therefore , we successfully complete primitive culture and subculture for human retinal pigment epithelium, and add Verapamil to induce the apoptosis in order to explore the effective concentration and time in inducing apoptosis. Thus it builds the theoretical foundation for the future research of the clinic application of verapamil to the proliferative disease.Material and Method1. Tissue source and preparationUnder sterile condition, we obtain the donator after the cornea transplantation from department of ophthalmology in the first affiliated hospital in China Medical University.2. The culture of Human RPEUnder sterile condition, using enzyme digestive method to isolate and complete plantation of humans RPE by 0. 25% trypsin to 24 - pore culture plate. The cells is transfered by 0. 25% trypsin after they become a confluent monolayer.3. Determination of the drug influnce to the proliferation of RPE through MTT colorimetric metrodThe cultured RPEs at density of 2 × 104/100μJ are plantated in 96 - pore culture plate. Adding verapamil at the concentration of 40mg/L,80mg/L,160mg/L,320mg/L to the cell in 96 -pore culture plate, after 24 hours, we measure the absorbency in 490nm wavelength on auto - enzyme - labeled meter using MTT colorimetric method . Statistics analysis is carried out to the data obtained.4. Verification of apoptosisWe choose three groups of the third generation RPE to comparethe apoptosis result. The first and the second group are added verapamil at the concentration of 80mg/L and 160mg/L respectively, and the third group is as a standard without verapamil. Three methods are used to observe the process. They are HE stain, Acridine Orange (AO) fluorescent stain and transmission electron microscopy. Adding PI stain to the RPEs which use verapamil within 12-72 hours, the specimens are measured by the flow cytometer. The apoptosis rate is obtained through calculating the quantity of cells in the apoptosis areaResultThe inhibiting rate is 13. 55% , 16. 71% , 42. 34% and 51. 01% respectively in the experiment of determination of the drug influence to the proliferation of RPE. . With the increase of the concentration of verapamil, the inhibiting rates rise. Based on the analysis of variance and t - test, it is verified that verapamil has strong inhibition on Human RPE ( HRPE) and an obvious dosage - dependent relationship. According to the results from the light microscopy, electron microscopy,fluorescence microscope and flow cytometer, the verapamil at the con...
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