FK506对高糖培养视网膜Müller细胞血管内皮生长因子表达的抑制作用

Inhibition of FK506 on the expression of vascular endothelial growth factor in retinal Müller cells cultured by high concentration glucose

背景 视网膜M＆#252;ller细胞可通过表达血管内皮生长因子（VEGF）参与糖尿病视网膜病变（DR）的病理过程.FK506可抑制实体肿瘤及实验性角膜新生血管中VEGF的表达,但其对视网膜M＆#252;ller细胞中VEGF的表达是否有抑制作用鲜见文献报道.目的 观察FK506对高糖培养的大鼠视网膜M＆#252;ller细胞表达血管内皮生长因子（VEGF）的影响.方法 将大鼠永生化视网膜Müller细胞系进行常规培养并将对数生长期的Müller细胞分别接种到96孔培养板中,分别在培养液中加入800.00、400.00、200.00、100.00、75.00、50.00、25.00、12.50和6.25 pg/ml FK506 100 μl/孔（细胞密度为1×10^4个/ml）,MTT比色法确定Müller细胞半数抑制浓度（IC50）的最适FK506质量浓度.将大鼠视网膜Müller细胞系分为正常对照组、FK506组、高糖组和高糖＋ FK506组,分别用高糖DMEM培养液（含50 mmol/L D-葡萄糖）和正常DMEM培养基（含5.5 mmol/L D-葡萄糖）进行培养,并分别在培养基中加入FK506,至质量浓度为75 pg/ml.ELISA法检测培养细胞上清液中VEGF的质量浓度;分别采用逆转录PCR（RT-PCR）和Western blot法检测各组大鼠视网膜Müller细胞中VEGF mRNA及其蛋白的相对表达量,分别以目的基因与内参β-actin吸光度（A）比值和灰度比值表示.结果 光学显微镜下正常对照组、FK506组、高糖组及高糖＋Fk506组培养12、24、48 h的大鼠视网膜Müller细胞均呈多角形,大小一致.致大鼠视网膜Müller细胞IC50的FK506质量浓度为75 pg/ml.正常对照组、FK506组、高糖组和高糖＋FK506组细胞上清中VEGF蛋白的质量浓度分别为（966.46±13.59） pg/ml、（1 059.42±67.43） pg/ml、（16 243.11±3 926.38）pg/ml和（9 467.25±1 525.56） pg/ml,总体差异有统计学意义（F=20.51,P=O.00）,其中高糖组细胞上清中VEGF的质量浓度明显高于正常对照组,差异有统计学意义（P=0.00）;高糖＋FK506组较高糖组细胞上清中VEGF的质量浓度降低,差异有统计学意义（P=0.02）;FK506组细胞上清中VEGF的质量浓度高于正常对照组,但差异无统计学意义（P=0.08）.4个组大鼠视网膜Müller细胞中VEGF mRNA及其蛋白相对表达量的总体差异均有统计学意义（F=126.06,P=0.00;F=5.44,P=0.01）,其中高糖组大鼠细胞中VEGF mRNA及其蛋白相对表达量均明显高于正常对照组,而高糖＋FK506组细胞中VEGF mRNA及其蛋白相对表达量均明显低于高糖组,差异均有统计学意义（P＜0.01）,FK506组细胞中VEGF mRNA及其蛋白的相对表达量分别为0.64±0.09和0.68±0.18,均低于正常对照组的0.84±0.07和0.75±0.03,但差异均无统计学意义（P=0.05、0.07）.结论 高糖条件下培养的Müller细胞中VEGF表达明显上调,FK506则可一定程度上抑制高糖培养的Müller细胞中VEGF的表达.

Background Retinal Muller cells participate in the pathological process of diabetic retinopathy （DR） through expressing vascular endothelial growth factor （VEGF）.It is reported that FK506 inhibits the expression of VEGF in solid tumors and experimental corneal neovascularization,but whether FK506 exerts its role on retinal Müller cells or not is still unclear.Objective This study aimed to investigate how FK506 affects the expression vascular endothelial growth factor （VEGF） in rat retinal Müller cells under the condition of high glucose.Methods Immortalized rat retinal Müller cell line was regularly cultivated and logarithmic phase of cells were incubated in 96-well plate with the cell density of 1×10^4/ml.Different concentrations of FK506 （800.00,400.00,200.00,100.00,75.00,50.00,25.00,12.50 and 6.25 pg/ml） （100 μl/well） were added into the culture medium to determine the half maximal inhibitory concentration （IC50） of FK506 by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT）.The cell lines were cultured with DMEM medium （containing D-glucose of 5.5 mmol/L） or high glucose DMEM （containing D-glucose of 50 mmol/L）,and 75 pg/ml FK506 were added into DMEM,respectively,and the cells were divided into the normal control group,FK506 group,high glucose culture group and high glucose ＋ FK506 group.ELISA was employed to assay the content of VEGF protein in the cell supernatant.The expressions of VEGF mRNA and protein in the cells were detected by reverse transcription PCR （RT-PCR） and Western blot,respectively.Results The cells grew well in the normal control group,FK506 group,high glucose culture group and high glucose＋FK506 group in 12,24 and 48 hours after culture with the polygon-like shape.The IC50 of FK506 was 75 pg/ml.The contents of FK506 in the cell supernatant were （966.46± 13.59） pg/ml,（1 059.42±67.43） pg/ml,（16 243.11 ±3 926.38） pg/ml and （9 467.25± 1 525.56） pg/ml in the normal control group,FK506 group,high glucose culture group and high glucose＋FK506 group,respectively,showing a significant difference among the four groups （F =20.51,P =0.00）.The VEGF levels in cell supernatant were significantly higher in the high glucose group than those of the normal group and the high FK506 group （P =0.00,P =0.02）,but no significant difference was found in the VEGF level in cell supernatant between the control group and FK506 group （P =0.08）.The expressions of VEGF mRNA and protein in the cells were significantly different among the four groups （F=126.06,P=0.00;F=5.44,P=0.01）,and the relative expressing values of VEGF mRNA and protein in the cells of the high glucose group were significantly higher than those of the control group and the high＋ FK506 group （all at P〈0.01）.The relative expressing values of VEGF mRNA and protein were 0.64±0.09 and 0.68±0.18 in the FK506 group,which were lower than those of the normal control group （0.84±0.07 and 0.75± 0.03）.However,no significant differences were seen between the two groups （P =0.05,0.07）.Conclusions The expression of VEGF in rat retinal Müller cells up-regulates under the high glucose condition.FK506 plays inhibitory effects on VEGF expression to certain extent in vitro.