雷帕霉素对人视网膜色素上皮细胞增生和凋亡的影响

The effects of rapamycin on the proliferation and apoptosis of human retinal pigment epithelium cells in vitro

背景雷帕霉素（RAPA）是哺乳动物RAPA靶蛋白（mTOR）特异性抑制剂，能有效抑制晶状体上皮细胞（LECs）及多种肿瘤细胞增生并具有诱导肿瘤细胞凋亡的作用，研究其对视网膜色素上皮（RPE）细胞是否具有类似作用对临床上预防和治疗增生性玻璃体视网膜病变（PVR）具有重要意义。目的研究RAPA对体外培养的人RPE细胞增生和凋亡的影响。方法对人RPE细胞（D407细胞株）进行体外传代培养，按照培养基中加入药物的不同将培养的细胞分为9组，空白对照组进行常规培养；二甲基亚砜（DMSO）对照组在培养基中加入质量分数0．1％。DMSO；实验各组将5、10、20、40、80、160、320nmol／LRAPA分别加入培养基中并分别培养12、24、48h。应用噻唑蓝（MTT）法检测各组吸光度（A490）值并计算各组人RPE细胞增生的抑制率，应用Hoechst染色法检测各组人RPE细胞的凋亡率，评价上述各浓度RAPA作用不同时间后对人RPE细胞增生和凋亡的影响。结果不同浓度RAPA组对人RPE细胞的抑制率随浓度的增高而明显升高，差异有统计学意义（F=484．451，P〈0．01），20～320nmol／LRAPA组在各时间点所测细胞增生抑制率均明显高于DMSO溶剂对照组，差异均有统计学意义（P〈0．01）。RAPA对细胞增生的抑制率随作用时间的延长明显增加，差异有统计学意义（F=232．262，P〈0．01），各浓度的RAPA作用24h和48h后细胞增生的抑制率均明显高于12h，差异均有统计学意义（P〈0．05）。与空白对照组及溶剂对照组比较，10nmol／LRAPA作用12、24、48h均有诱导细胞凋亡的作用，差异均有统计学意义（P〈0．05）；20～320nmol／LRAPA组细胞凋亡率明显增加，差异有统计学意义（P〈0．叭）。各浓度RAPA组随作用时间的延长人RPE细胞的凋亡率明显增加（F=625．584，P〈0．01）。Hoechst33258染色可见凋亡细胞核碎裂并呈块状致密浓染，染色质固缩。结论RAPA以浓度和时间依赖的方式抑制体外培养的人RPE细胞增生并诱导其凋亡。

Background Rapamycin （RAPA） is a specific inhibitor of the mammalian target of rapamycin （mTOR）. Researches showed that RAPA inhibits the proliferation of lens epithelium cells （LECs） and tumor cells and induces apoptosis of tumor cells. To investigate whether rapamycin has the inhibitory effect on retinal pigment epithelium （RPE） cells is very important for the prevention and management of proliferative vitreoretinopathy （ PVR）. Objective This study was to investigate the effects of RAPA on the proliferation and apoptosis of human RPE cells in vitro. Methods Human RPE cells （D407 strain） were cultured and passaged and then divided into regular culture group （blank control group） , DMSO control group （0.1%o DMSO ＋regular culture） , and different concentrations RAPA-treatment groups （ 5,10,20,40,80,160,320 nmol/L）. The proliferation （ A490 ） of human RPE cells was detected using MTT,and the inhibitory rates of RAPA on the proliferation of RPE cells were calculated and compared among different groups at 12,24 and 48 hours. The apoptosis rates of the cells were analyzed among various groups by Hoechst staining after 12,24,48 hours. Results The inhibitory rates of RAPA on RPE cells were significantly different among various groups （F=484. 451 ,P〈0.01 ） and evidently elevated in 20-320 nmol/L RAPA groups compared with DMSO control group （ P 〈 0.01 ）. The inhibition of RAPA on the cells was considerably enhanced as the lapse of time （ F = 232. 262, P〈0.01 ） with more dominant effects in 24 and 48 hours compared to 12 hours after addition of RAPA （P〈0. 05-0. 01 ）. Compared with blank control group and DMSO control group, the apoptotic rates of the cells were evidently increased in 12,24,48 hours in 10 nmol/L RAPA group （ all P〈0.05 ） , and higher cellular apoptotic rates were found in 20-320 nmol/L RAPA groups （ all P〈0.01 ）. The alteration of cellular apoptotic rate showed a gradually incremental trend as the acting time of RAPA （F= 625. 584 ,P〈0.01 ）. Karyorrhexis and mass-like density staining and chromatin substance were seen in RPE cells under the fluorescence microscope in ≥ 10 nmoi/L RAPA groups. Conclusions RAPA suppresses the proliferation and induces the apoptosis of human RPE cells in concentration-and time-dependent manner in vitro.