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，Q72CF=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。

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(Q_48AB=44.0427, Q_48AC=36.4701, Q_48BC=27.7953, all P> 0.05), the remaining pairwise comparisons were statistically significant difference between groups(Q_48CD=11.0715, Q_48AD=14.0794, Q_48AF=12.2964, Q_48BD=23.4698, Q_48BF=12.7016, Q_48DF=10.2013, all P<0.05; Q_48CF=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(Q_72AB=27.7338, Q_72AC=25.0054, Q_72BC=33.3797, all P>0.05), the rest of the group differences were statistically significant(Q_72AD=13.4793, Q_72BD=12.7546, both P<0.05; Q_72CD=7.3743, Q_72CF=8.3465, Q_72AF=4.7455, Q_72BF=3.9471, Q_72DF=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, Q_48AB=31.1704, Q_48AC=33.5947, Q_48BC=29.3968, Q_48AD=30.4097, Q_48BD=28.8164, both P>0.05; Q_48CD=12.5032, Q_48AF=11.7531, Q_48BF=14.1076, Q_48DF=13.9372, both P<0.05; Q_48CF=7.0953, P<0.01. After 72h culture, pairwise comparison among five groups, Q_72AB=26.3392, Q_72AC=24.9142, Q_72BC=30.2976, both P>0.05, the rest of the group differences were statistically significant(where Q_72AD=14.1983, Q_72BD=12.9356, both P<0.05; Q_72CD=6.8752, Q_72CF=7.6745, Q_72AF=5.0545, Q_72BF=4.0741, Q_72DF=3.8876, both P<0.01).

CONCLUSION: Best glucose concentration and the time is 25 mmol/L and 48h, for RVEC high sugar model.