雷帕霉素对体外培养的人视网膜色素上皮细胞增殖的影响

发布时间：2018-04-30 04:25

本文选题：增生性玻璃体视网膜病变 + 视网膜色素上皮细胞　；参考：《吉林大学》2012年硕士论文

【摘要】：研究表明,视网膜色素上皮(retinal pigment epithelium, RPE)细胞是增生性玻璃体视网膜病变(proliferation vitreoretinopathy, PVR)的增殖膜中的主要细胞来源,因此增殖性玻璃体视网膜病变的研究和防治多围绕探讨RPE细胞的异常行为和抑制RPE细胞及其他相关细胞的增殖。雷帕霉素(rapamycin, RAPA)是一种新型的大环内酯类免疫抑制剂,眼科方面已有研究应用于动物及体外细胞培养药物实验。目的：本研究是在体外培养人视网膜色素上皮(human retinal pigment epithelium, hRPE)细胞的基础之上,探讨一定范围浓度的RAPA对体外培养的hRPE细胞形态、增殖等生物学活性的影响。方法：复苏并体外培养hRPE细胞并分为不同浓度雷帕霉素实验组(Ong/ml,5ng/ml,10ng/ml,20ng/ml,40ng/ml,80ng/ml)。显微镜下观察各组细胞及药物不同作用时间下,细胞的增殖状态、倍增时间、细胞活力、生长曲线及四唑盐(MTT)比色法检RAPA对hRPE细胞增殖的影响。结果：复苏后细胞活力良好的hRPE细胞株,雷帕霉素在实验范围内抑制hRPE细胞的增殖。镜下可见随雷帕霉素浓度增加,细胞数目逐渐减少。经80ng/ml雷帕霉素作用72小时后部分细胞变形崩解,偶见细胞浮起。对照组细胞分裂时间为1.83天,随RAPA药物浓度增加细胞分裂时间随之延长。生长曲线显示随着雷帕霉素浓度的增加,作用时间的延长,hRPE细胞的生长有明显的抑制作用,并呈剂量时间效应关系。通过生长曲线可见对照组细胞增殖旺盛,各项生长指标均高于用药组。台盼兰细胞活力测定结果为不同浓度雷帕霉素(Ong/ml,5ng/ml,1Ong/ml,20ng/ml,40ng/ml,80ng/ml)细胞活力分别为92.3%、89.3%、88.7%、87.7%、86.0%、82.6%。MTT比色法观察细胞生长基本规律结果示：各浓度药物组作用24小时后与对照组比较计算得抑制率,分别为8.70%、22.06%、40.69%、42.31%、43.12%；72小时时分别为12.57%、26.35%、42.23%44.15%、47.12%。结论： 1雷帕霉素对体外培养的hRPE细胞增殖具有有效抑制作用,其效应在一定范围内呈浓度/时间依赖性。10～80ng/ml浓度范围内,RAPA明显抑制hRPE细胞增殖(P0.01)；但在20～80ng/ml浓度范围内,各组间抑制作用无显著性差异(P0.05)。 2雷帕霉素延长hRPE细胞倍增时间,降低体外培养的hRPE细胞活性。 3雷帕霉素在80ng/ml时细胞抑制率近50%。 4雷帕霉素可能为临床预防和治疗PVR提供新思路,提升其在眼科的应用价值。
[Abstract]:The results show that retinal pigment epithelium (RPE) cells are the main cell sources in proliferative membrane of proliferative vitreoretinopathy (PVR) of proliferative vitreoretinopathy (PVR). Therefore, the study and prevention of proliferative vitreoretinopathy focus on the study of abnormal behavior of RPE cells and inhibition of proliferation of RPE cells and other related cells. Rapamycin (rapamycin) is a new type of macrolide immunosuppressant, which has been used in animal and in vitro cell culture experiments in ophthalmology. Objective: Based on the culture of human retinal pigment epithelium (RPE) retinal pigment epithelium, hRPE) cells in vitro, the effects of certain concentration of RAPA on the morphology and proliferation of hRPE cells in vitro were investigated. Methods: HRPE cells were resuscitated and cultured in vitro and divided into different concentrations of rapamycin experimental group: 5 ng / ml 10 ng / ml 10 ng / ml 20 ng / ml 40 ng / ml / ml 80 ng / ml / ml. The effects of RAPA on the proliferation of hRPE cells were observed under microscope. The proliferation state, doubling time, cell viability, growth curve and tetrazolium trioxide (MTT) colorimetric assay were used to detect the effect of RAPA on the proliferation of hRPE cells. Results: After resuscitation, rapamycin inhibited the proliferation of hRPE cells. Microscopically, the number of cells decreased with the increase of rapamycin concentration. After 72 hours of treatment with 80ng/ml rapamycin, some of the cells were deformed and disintegrated, and occasionally the cells floated. The cell division time of the control group was 1.83 days, and the cell division time was prolonged with the increase of RAPA concentration. The growth curve showed that with the increase of rapamycin concentration, the growth of hRPE cells was significantly inhibited with the prolongation of the action time, and showed a dose-time effect. The growth curve showed that the growth index of the control group was higher than that of the drug group. Trypan blue cell viability assay showed that the cell viability of different concentrations of rapamycin Ong / ml 5 ng / ml 1 Ong / ml + 20 ng / ml 20 ng / ml + 20 ng / ml + 40ng / ml + 80ng / ml) was 92.3% and 89.3% respectively. The cell growth was observed by MTT colorimetric method. The results showed that the inhibition rate was calculated after 24 hours of treatment in each concentration group as compared with the control group. 42.31 and 43.12 for 72 hours, 12.57 and 26.35, 42.23D.15 and 47.12. respectively, with 8.70 and 22.06 and 40.69, and 42.31 and 43.12, respectively, with 12.57 and 26.35, and 42.23D.15 and 47.12, respectively. Conclusion: 1 rapamycin had an effective inhibitory effect on the proliferation of hRPE cells cultured in vitro, and its effect was in a dose-dependent / time-dependent manner. Rapa significantly inhibited the proliferation of hRPE cells in a concentration range of 80 ng / ml, but in the range of 20~80ng/ml concentration, rapamycin inhibited the proliferation of hRPE cells in a dose-dependent manner, but in the range of 20~80ng/ml concentration, rapamycin inhibited the proliferation of hRPE cells in a dose-dependent manner. There was no significant difference in the inhibitory effect among the three groups (P 0.05). 2 rapamycin prolonged the doubling time of hRPE cells and decreased the activity of hRPE cells cultured in vitro. 3 the inhibitory rate of rapamycin on 80ng/ml was nearly 50%. Rapamycin may provide a new idea for clinical prevention and treatment of PVR, and enhance its application value in ophthalmology.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1

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