缺氧和高糖对视网膜色素上皮细胞增生及VEGF、TGF-β1表达的影响

Effect of hypoxia and high glucose on proliferation of retinal pigment epithelial cells and expression of VEGF and TGF-β1

目的探究缺氧、高糖环境对体外培养的视网膜色素上皮(retinal pigment epithelium,RPE)细胞生长、增生及血管内皮生长因子(vascular endothelial growth factor,VEGF)、转化生长因子-β1(transforming growth factor-β1,TGF-β1)表达变化的影响。方法采用酶消化法进行C57小鼠RPE细胞的体外原代培养。通过组织形态学观察和免疫化学染色对培养的RPE细胞进行鉴定。实验缺氧组:在DMEM培养液中加入CoCl2,使其浓度分别为50μmol.L-1、100μmol.L-1、200μmol.L-1;实验高糖组:在DMEM培养液中加入D-葡萄糖,使其浓度分别为15mmol.L-1、30mmol.L-1、45mmol.L-1。分别向培养的RPE细胞中加入上述培养液,共6组,每组3份;正常对照组加入含5.5mmol.L-1葡萄糖、不含CoCl2的培养液。观察各组细胞在不同培养条件下的活力、倍增时间及增生情况。采用ELISA法检测各组细胞在培养不同时间后(24h、48h、72h)上清液中VEGF及TGF-β1表达量的变化。结果原代培养及传代早期的RPE细胞生长较为旺盛;在缺氧和高糖培养条件下所培养的细胞活力和分裂次数均较正常对照组有所降低或减少,而倍增时间较正常对照组延长。在缺氧和高糖培养条件下培养24h、48h、72h后,细胞上清液中VEGF和TGF-β1表达量均较正常对照组高(P<0.05);当CoCl2浓度为100μmol.L-1和D-葡萄糖浓度为30mmol.L-1时,VEGF和TGF-β1在24h、48h、72h表达均高于其他浓度,缺氧状态下48h表达量分别为(43.28±0.88)ng.L-1和(8.90±1.38)ng.L-1,高糖状态下分别为(39.76±0.56)ng.L-1和(8.81±0.92)ng.L-1;缺氧组2种细胞因子表达量在48h达到高峰,而高糖组在72h达到高峰。结论应用酶联合消化法可获得纯度较高的体外C57小鼠培养RPE细胞;缺氧和高糖状态可对RPE细胞增生起到明显抑制作用,但能够明显上调RPE细胞的VEGF、TGF-β1表达量。

Objective To investigate the cell growth and proliferation of retinal pigment epithelium （RPE） and the expression of vascular endothelial growth factor （VEGF） and transforming growth factor-β1 （TGF-β1） under hypoxia and high glucose environment. Methods RPE cells of C57 mice were primary cultured in vitro with enzyme digestion. The cultured RPE cells were identified with histomorphological observation and immunocytochemical staining. In experimental hypoxia groups, the concentrations of CoCl2 added were 50 μmol · L^-1, 100 μmol · L^-1 ,200 μmol · L^-1 in DMEM culture fluid. In experimental high glucose groups ,the concentrations of D-glucose added were 15 mmol · L^-1,30 mmol · L^-1 ,45 mmol · L^-1 in DMEM culture fluid. Each of above-mentioned culture fluid was added to cultured RPE cells, and there was 6 groups, triplicate for each group. The culture fluid of normal control group contained 5.5 mmol · L^-1 glucose, but without CoCl2. The vital force, doubling times and proliferation were observed. The expressive changes of VEGF, TGF-β1 in the supernanant at different time （24 hours,48 hours,72 hours） were also examined by ELISA. Results The primary and the first generation RPE cells grew well;Cells cultured in hypoxia or high glucose medium reduced in vital force and population doubling, but lengthened in doubling time;Expression of VEGF and TGF-β1 in the hypoxia and high glucose groups were higher than those in the control group after 24 hours,48 hours,72 hours（P 〈0.05） ;The peaks appeared in 100mmol · L^-1 CoCl2 group[ （43.28±0. 88）ng · L^-1 and （8.90±1.38） ng · L^-1 respectively after 48 hours ] and 30 mmol· L^-1 high glucose group [ （ 39. 76±0.56） ng· L^-1 and （ 8.81± 0.92 ）ng· L^-1 respectively after 48 hours ] at different time, comparing with other concentrations. The peak of cellular factors expression appeared at 48 hours in hypoxia group, and at 72 hours in high glucose group. Conelusions Purified RPE cells are obtained by enzyme digestion;Both hypoxia and high glucose environments can obviously inhibit the proliferation of the RPE cells, but increase the expressions of VEGF and TGF-β1.