论文部分内容阅读
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 effects of hypoxia and high glucose on cell growth, proliferation and the expression of vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1) in cultured retinal pigment epithelium (RPE) β1, TGF-β1) expression changes. Methods The primary culture of RPE cells in C57 mice was performed by enzyme digestion. The cultured RPE cells were identified by histomorphological observation and immunochemical staining. In the experimental hypoxia group, CoCl2 was added into the DMEM medium to make them concentration of 50μmol.L-1, 100μmol.L-1,200μmol.L-1, respectively. Experimental high glucose group: D-glucose , So that its concentration was 15mmol.L-1, 30mmol.L-1, 45mmol.L-1. Each of the above culture broths was added to the cultured RPE cells in a total of 6 groups of 3 parts each. The normal control group was supplemented with 5.5 mmol.L-1 glucose and CoCl2-free medium. The activity, doubling time and hyperplasia of cells in each group were observed under different culture conditions. The expression of VEGF and TGF-β1 in the supernatant of each group were detected by ELISA at different time points (24h, 48h, 72h). Results The primary culture and early passage of RPE cells grew more vigorously. The cell viability and number of division of cells cultured in hypoxia and high glucose medium were lower or lower than those in normal control group, while the doubling time was longer than that in normal control group . The expression of VEGF and TGF-β1 in the cell supernatant was higher than that in the normal control group after hypoxia and high glucose culture for 24h, 48h and 72h (P <0.05). When the concentration of CoCl2 was 100μmol·L-1 And D-glucose 30 mmol·L-1, the expression of VEGF and TGF-β1 at 24 h, 48 h and 72 h were all higher than those at other concentrations. The expression of VEGF and TGF-β1 at 48 h after hypoxia were (43.28 ± 0.88) ng.L-1 And (8.90 ± 1.38) ng.L-1, respectively, and (39.76 ± 0.56) ng.L-1 and (8.81 ± 0.92) ng.L-1 in high glucose group. The expression of the two cytokines in hypoxia group was 48h reached the peak, while the high glucose group reached a peak at 72h. CONCLUSION: Purified RPE cells can be obtained by enzymatic digestion in vitro. Hypoxia and high glucose can significantly inhibit the proliferation of RPE cells, but significantly upregulate the expression of VEGF and TGF-β1 in RPE cells the amount.