视网膜血管内皮细胞高糖模型糖浓度及培养时间的筛选

Filtering glucose concentration and culture time of Wistar rat retinal vascular endothelial cells in high glucose model

目的:筛选Wistar大鼠视网膜血管内皮细胞(retinal vascular endothelial cells,RVEC)高糖模型糖浓度及培养时间。方法:取出生1~3d的Wistar大鼠,分离RVEC进行原代培养,Ⅷ因子抗体免疫组化鉴定细胞;设立葡萄糖浓度0mmol/L为正常对照组(C组),按不同葡萄糖浓度依次分为10mmol/L(A组)、15mmol/L(B组)、25mmol/L(D组)、35mmol/L(F组),分别在24、48、72h的3个时间点用MTT法检测各组细胞OD值,流式细胞仪检测各组细胞凋亡率。结果:RVEC纯度达94%;MTT法检测不同浓度葡萄糖对Wistar大鼠RVEC的影响:培养24h,各组的OD值比较差异无统计学意义(F=0.42,P=0.7411);培养48h,五组OD值比较差异有统计学意义(F=45.2,P=0.000),五组间两两比较,除A组、B组、C组间差异无统计学意义外(Q_(48AB)=44.0427,Q_(48AC)=36.4701,Q_(48BC)=27.7953,均P>0.05),其余各组两两比较差异均有统计学意义(其中Q_(48CD)=11.0715,Q_(48AD)=14.0794,Q_(48AF)=12.2964,Q_(48BD)=23.4698,Q_(48BF)=12.7016,Q_(48DF)=10.2013,均P<0.05;Q_(48CF)=6.4701,P<0.01)。培养72h后,五组OD值比较差异有统计学意义(F=37.46,P=0.000),五组间两两比较,同样A、B、C三组间差异无统计学意义(Q_(72AB)=27.7338,Q_(72AC)=25.0054,Q_(72BC)=33.3797,均P>0.05),其余各组差异均有统计学意义(其中Q_(72AD)=13.4793,Q_(72BD)=12.7546,均P<0.05;Q_(72CD)=7.3743,Q_(72CF)=8.3465,Q_(72AF)=4.7455,Q_(72BF)=3.9471,Q_(72DF)=3.2649,均P<0.01)。不同浓度葡萄糖在不同时间点对RVEC凋亡率的影响:培养24h后各组间差异无统计学意义(P>0.05)。培养48h后,五组间两两比较,Q_(48AB)=31.1704,Q_(48AC)=33.5947,Q_(48BC)=29.3968,Q_(48AD)=30.4097,Q_(48BD)=28.8164,均P>0.05;Q_(48CD)=12.5032,Q_(48AF)=11.7531,Q_(48BF)=14.1076,Q_(48DF)=13.9372,均P<0.05;Q_(48CF)=7.0953,P<0.01。培养72h后,五组间两两比较,Q_(72AB)=26.3392,Q_(72AC)=24.9142,Q_(72BC)=30.2976,均P>0.05,其余各组差异均有统计学意义(其中Q_(72AD)=14.1983,Q_(72BD)=12.9356,均P<0.05;Q_(72CD)=6.8752,Q_(72CF)=7.6745,Q_(72AF)=5.0545,Q_(72BF)=4.0741,Q_(72DF)=3.8876,均P<0.01)。结论:葡萄糖浓度为25mmol/L培养48h是RVEC高糖模型最佳葡萄糖浓度及干预时间。

AIM： To filter glucose concentration and culture time of Wistar rat retinal vascular endothelial cells（RVEC）in high glucose model. METHODS： Primary culture of RVEC of Wistar rats with 1-3d of birth,Ⅷ factor antibody immunohistochemistry to identify the cells; Establishment 0mmol/L glucose as a control group（group C）, at different concentrations of glucose intervention in turn divided into 10mmol/L（group A）, 15mmol/L（group B）, 25mmol/L（group D）, 35mmol/L（group F）, MTT assay was used to detect cell OD values of each group and each group was detected by flow cytometry apoptosis rate were at three time points： 24,48,72h.RESULTS： RVEC purity was 94%. Effect of different concentrations of glucose in Wistar rat RVEC by MTT assay： after 24h culture, OD value of experimental group decreased, but the difference was not statistically significant（F=0.42, P=0.7411）; after 48h culture, OD value of the difference between the groups was statistically significant（F=45.2, P=0.000）, pairwise comparison between the five groups, except among groups A, B, C group no difference was not statistically significant external（Q48AB=44.0427, Q48AC=36.4701, Q48BC=27.7953, all P〉 0.05）, the remaining pairwise comparisons were statistically significant difference between groups（Q48CD=11.0715, Q48AD=14.0794, Q48AF=12.2964, Q48BD=23.4698, Q48BF=12.7016, Q48DF=10.2013, all P〈0.05; Q48CF=6.4701, P〈0.01）. After 72h culture, OD value of each group compared to the difference was statistically significant（F=37.46, P=0.000）, pairwise comparisons among five groups, the same A, B, C no difference among the three groups was not statistically significant（Q72AB=27.7338, Q72AC=25.0054, Q72BC=33.3797, all P〉0.05）, the rest of the group differences were statistically significant（Q72AD=13.4793, Q72BD=12.7546, both P〈0.05; Q72CD=7.3743, Q72CF=8.3465, Q72AF=4.7455, Q72BF=3.9471, Q72DF=3.2649, all P〈0.01）. Effect of different concentrations of glucose on RVEC apoptosis rate： after 24h culture, Differences between groups was not statistically significant（P〉0.05）. After 48h culture, pairwise comparison among five groups, Q48AB=31.1704, Q48AC=33.5947, Q48BC=29.3968, Q48AD=30.4097, Q48BD=28.8164, both P〉0.05; Q48CD=12.5032, Q48AF=11.7531, Q48BF=14.1076, Q48DF=13.9372, both P〈0.05; Q48CF=7.0953, P〈0.01. After 72h culture, pairwise comparison among five groups, Q72AB=26.3392, Q72AC=24.9142, Q72BC=30.2976, both P〉0.05, the rest of the group differences were statistically significant（where Q72AD=14.1983, Q72BD=12.9356, both P〈0.05; Q72CD=6.8752, Q72CF=7.6745, Q72AF=5.0545, Q72BF=4.0741, Q72DF=3.8876, both P〈0.01）. CONCLUSION： Best glucose concentration and the time is 25 mmol/L and 48h, for RVEC high sugar model.