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目的探讨替德肝素(tedelparin)在抑制冷冻后的人视网膜色素上皮(humanretinapigmentepithelium,hRPE)细胞分泌肝细胞生长因子(humanhepatocytegrowthfactor,HGF)和生长中的作用。方法体外培养的RPE细胞在-80℃下进行冷冻,冷冻时间分为0、15和60s,随后继续体外培养(体外实验组)和注入正常兔眼玻璃体(体内实验组)并在第6天时取出一些兔眼的玻璃体液加入到正常RPE细胞培养液中孵育48h;每实验组再分为两个亚组替德肝素治疗(终浓度25μl/ml)组和未治疗组,在3d、6d时收集细胞培养上清和玻璃体样本,ELISA法测定HGF含量,MTT法测定48h后RPE细胞的增殖状态。结果在体外实验组,比较未冷冻组,冷冻后的RPE细胞随着冷冻时间延长HGF分泌水平增加(F=2736,P<001),替德肝素干预组HGF分泌水平下降(F=17950,P<001)。在体内实验组(兔玻璃体)随着冷冻时间延长HGF浓度较对照组明显增高(F=624,P<001),当玻璃体液(冷冻15s和60s,第6天)加入到正常RPE细胞培养液48h后刺激细胞增殖(P<001),在替德肝素干预组,细胞增殖明显减弱(F=4490,P<005)。结论冷冻可刺激RPE细胞在体外和体内玻璃体环境中HGF的高分泌,且随着冷冻时间增加更为显著。替德肝素可降低冷冻后RPE细胞分泌HGF水平和抑制促生长环境中的RPE细胞生长,具有预防PVR的作用。
Objective To investigate the role of tedelparin in inhibiting human hepatocyte growth factor (hGF) and its growth in frozen human retinal pigment epithelium cells (hRPE). Methods RPE cells cultured in vitro were frozen at -80 ° C and were frozen at 0, 15, and 60 s and then continued in vitro (in vitro) and injected into normal rabbit vitreous (in vivo experiments) and removed on day 6 Some rabbit eyes vitreous fluid was added to the normal RPE cell culture medium incubated for 48h; each experimental group was further divided into two subgroups of adeflunin treatment (final concentration 25μl / ml) and untreated group collected at 3d, 6d Cell culture supernatants and vitreous samples were collected. HGF content was determined by ELISA. The proliferation of RPE cells was measured by MTT assay. Results Compared with the non-freezing group, the levels of HGF secretion increased (F = 2736, P <001) and the secretion of HGF in frozen-thawed RPE cells decreased after freezing (F = 17950, P <001). HGF concentration in the experimental group (rabbit vitreous) increased significantly as the freezing time prolonged (F = 624, P <001). When the vitreous humor (frozen for 15s and 60s, on the 6th day) was added to the normal RPE cell culture Cell proliferation was stimulated at 48h (P <001). Cell proliferation was significantly attenuated in the intervention group (F = 4490, P <005). Conclusion Cryopreservation can stimulate the secretion of HGF in RPE cells in vitro and in vitreous environment, and it is more significant with the increase of freezing time. Tidulokarin can reduce the level of HGF secreted by RPE cells after cryopreservation and inhibit the growth of RPE cells in a growth-promoting environment, which has the effect of preventing PVR.