中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
11期
998-1003
,共6页
夏蔚%夏静%张晓峰%钟蕾%孙正太%王英明
夏蔚%夏靜%張曉峰%鐘蕾%孫正太%王英明
하위%하정%장효봉%종뢰%손정태%왕영명
抗生素/FK506%视网膜%胶质细胞/Müller细胞%血管内皮生长因子%高糖%细胞培养
抗生素/FK506%視網膜%膠質細胞/Müller細胞%血管內皮生長因子%高糖%細胞培養
항생소/FK506%시망막%효질세포/Müller세포%혈관내피생장인자%고당%세포배양
Antibiotic agent/FK506%Retina%Glial cell/Müller cell%Vascular growth factor%High glucose condition%Cell culture
背景 视网膜Müller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Müller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Müller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×l04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.
揹景 視網膜Müller細胞可通過錶達血管內皮生長因子(VEGF)參與糖尿病視網膜病變(DR)的病理過程.FK506可抑製實體腫瘤及實驗性角膜新生血管中VEGF的錶達,但其對視網膜Müller細胞中VEGF的錶達是否有抑製作用鮮見文獻報道.目的 觀察FK506對高糖培養的大鼠視網膜Müller細胞錶達血管內皮生長因子(VEGF)的影響.方法 將大鼠永生化視網膜Müller細胞繫進行常規培養併將對數生長期的Müller細胞分彆接種到96孔培養闆中,分彆在培養液中加入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(細胞密度為1×l04箇/ml),MTT比色法確定Müller細胞半數抑製濃度(IC50)的最適FK506質量濃度.將大鼠視網膜Müller細胞繫分為正常對照組、FK506組、高糖組和高糖+ FK506組,分彆用高糖DMEM培養液(含50 mmol/L D-葡萄糖)和正常DMEM培養基(含5.5 mmol/L D-葡萄糖)進行培養,併分彆在培養基中加入FK506,至質量濃度為75 pg/ml.ELISA法檢測培養細胞上清液中VEGF的質量濃度;分彆採用逆轉錄PCR(RT-PCR)和Western blot法檢測各組大鼠視網膜Müller細胞中VEGF mRNA及其蛋白的相對錶達量,分彆以目的基因與內參β-actin吸光度(A)比值和灰度比值錶示.結果 光學顯微鏡下正常對照組、FK506組、高糖組及高糖+Fk506組培養12、24、48 h的大鼠視網膜Müller細胞均呈多角形,大小一緻.緻大鼠視網膜Müller細胞IC50的FK506質量濃度為75 pg/ml.正常對照組、FK506組、高糖組和高糖+FK506組細胞上清中VEGF蛋白的質量濃度分彆為(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,總體差異有統計學意義(F=20.51,P=O.00),其中高糖組細胞上清中VEGF的質量濃度明顯高于正常對照組,差異有統計學意義(P=0.00);高糖+FK506組較高糖組細胞上清中VEGF的質量濃度降低,差異有統計學意義(P=0.02);FK506組細胞上清中VEGF的質量濃度高于正常對照組,但差異無統計學意義(P=0.08).4箇組大鼠視網膜Müller細胞中VEGF mRNA及其蛋白相對錶達量的總體差異均有統計學意義(F=126.06,P=0.00;F=5.44,P=0.01),其中高糖組大鼠細胞中VEGF mRNA及其蛋白相對錶達量均明顯高于正常對照組,而高糖+FK506組細胞中VEGF mRNA及其蛋白相對錶達量均明顯低于高糖組,差異均有統計學意義(P＜0.01),FK506組細胞中VEGF mRNA及其蛋白的相對錶達量分彆為0.64±0.09和0.68±0.18,均低于正常對照組的0.84±0.07和0.75±0.03,但差異均無統計學意義(P=0.05、0.07).結論 高糖條件下培養的Müller細胞中VEGF錶達明顯上調,FK506則可一定程度上抑製高糖培養的Müller細胞中VEGF的錶達.
배경 시망막Müller세포가통과표체혈관내피생장인자(VEGF)삼여당뇨병시망막병변(DR)적병리과정.FK506가억제실체종류급실험성각막신생혈관중VEGF적표체,단기대시망막Müller세포중VEGF적표체시부유억제작용선견문헌보도.목적 관찰FK506대고당배양적대서시망막Müller세포표체혈관내피생장인자(VEGF)적영향.방법 장대서영생화시망막Müller세포계진행상규배양병장대수생장기적Müller세포분별접충도96공배양판중,분별재배양액중가입800.00、400.00、200.00、100.00、75.00、50.00、25.00、12.50화6.25 pg/ml FK506 100 μl/공(세포밀도위1×l04개/ml),MTT비색법학정Müller세포반수억제농도(IC50)적최괄FK506질량농도.장대서시망막Müller세포계분위정상대조조、FK506조、고당조화고당+ FK506조,분별용고당DMEM배양액(함50 mmol/L D-포도당)화정상DMEM배양기(함5.5 mmol/L D-포도당)진행배양,병분별재배양기중가입FK506,지질량농도위75 pg/ml.ELISA법검측배양세포상청액중VEGF적질량농도;분별채용역전록PCR(RT-PCR)화Western blot법검측각조대서시망막Müller세포중VEGF mRNA급기단백적상대표체량,분별이목적기인여내삼β-actin흡광도(A)비치화회도비치표시.결과 광학현미경하정상대조조、FK506조、고당조급고당+Fk506조배양12、24、48 h적대서시망막Müller세포균정다각형,대소일치.치대서시망막Müller세포IC50적FK506질량농도위75 pg/ml.정상대조조、FK506조、고당조화고당+FK506조세포상청중VEGF단백적질량농도분별위(966.46±13.59) pg/ml、(1 059.42±67.43) pg/ml、(16 243.11±3 926.38)pg/ml화(9 467.25±1 525.56) pg/ml,총체차이유통계학의의(F=20.51,P=O.00),기중고당조세포상청중VEGF적질량농도명현고우정상대조조,차이유통계학의의(P=0.00);고당+FK506조교고당조세포상청중VEGF적질량농도강저,차이유통계학의의(P=0.02);FK506조세포상청중VEGF적질량농도고우정상대조조,단차이무통계학의의(P=0.08).4개조대서시망막Müller세포중VEGF mRNA급기단백상대표체량적총체차이균유통계학의의(F=126.06,P=0.00;F=5.44,P=0.01),기중고당조대서세포중VEGF mRNA급기단백상대표체량균명현고우정상대조조,이고당+FK506조세포중VEGF mRNA급기단백상대표체량균명현저우고당조,차이균유통계학의의(P＜0.01),FK506조세포중VEGF mRNA급기단백적상대표체량분별위0.64±0.09화0.68±0.18,균저우정상대조조적0.84±0.07화0.75±0.03,단차이균무통계학의의(P=0.05、0.07).결론 고당조건하배양적Müller세포중VEGF표체명현상조,FK506칙가일정정도상억제고당배양적Müller세포중VEGF적표체.
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 × 104/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.
