高糖及高胰岛素对小鼠骨髓内皮前体细胞增殖及功能的影响

Effects of high levels of glucose and insulin on proliferation,apoptosis and cell function of mouse endothelial progenitor cells

目的研究高糖及高胰岛素对骨髓内皮前体细胞(EPCs)增殖、凋亡及分泌功能的影响。方法通过密度梯度离心法从小鼠(BALB/c)的骨髓中分离出EPCs,将细胞分为4组,A组为对照组(葡萄糖浓度5.5mmol/L),B、C组葡萄糖浓度分别为11.0 mmol/L、22.0 mmol/L,D组(葡萄糖浓度22.0 mmol/L+胰岛素25U/L)。利用四甲基偶氮唑盐(MTT)法检测细胞的增殖能力,流式细胞仪检测细胞凋亡,硝酸还原酶法测定细胞培养液中一氧化氮(NO)、一氧化氮合酶(NOS)的含量。结果骨髓源性EPCs的增殖能力与A组(1.31±0.21)比较,B、C、D组(B组0.93±0.27,C组0.78±0.31,D组0.57±0.24)均明显降低,差异有统计学意义(均为P<0.05);B、C、D组间比较差异无统计学意义(均为P>0.05)。实验5 d后,B组的凋亡率为(17.95%±0.38%)、C组的凋亡率为(32.5%±0.63%)、D组的凋亡率为(31.48%±1.26%),3组的凋亡率均较A组的凋亡率(15.75%±0.60%)增高;C组及D组凋亡率均较B组增加,差异均有统计学意义(均为P<0.01)。细胞分泌NO、总NOS(TNOS)显著减少、而诱导型NOS(iNOS)分泌显著增加(均为P<0.01)。结论高糖及高胰岛素引起的EPCs增殖能力降低,促使细胞凋亡,并损害细胞的分泌功能;高胰岛素对高糖引起的EPCs损害并未改善。

Objective To study the effects of high levels of glucose and insulin on proliferation, apoptosis and cell function of endothelial progenitor cells （EPCs） in mice. Methods EPCs were isolated from bone-marrow of mice by density gradient centrifugation method and were divided into four groups： Group A （control group, glucose concentration 5.5 mmol/L）, group B （glucose concentration 11.0 mmol/L）, group C （glucose concentration 22.0 mmol/L）, and group D （glucose concentration 22.0 mmol/L plus insulin 25 U/L）. Proliferation of EPCs was determined by MTT assay and apoptosis by flow cytometry. NO and NOS （TNOS,iNOS） in cell culture medium were measured. Results Proliferation ability of EPCs in group B,C and D was similar but significantly decreased compared to group A [ group A （ 1.31 -＋0. 21 ）, group B （0. 93 ±0. 27） ,group C （0. 78 ± 0. 31 ） and group D （0. 57± 0. 24）, all P 〈 0. 05 ]. After 5 days, apoptosis rates of EPCs in group B, C and D were significantly increased compared to group A [group A （15.75 ±0. 60）%, group B （ 17.95 ± 0. 38 ） %, group C （ 32. 5 ± 0. 63 ） % and group D （ 31.48 ± 1.26 ） %, all P 〈 0. 01 ]. Apoptosis rates were higher in group C and D than in group B. Releases of NO and TNOS in group B, C and D were decreased and iNOS increased significantly compared to group A （ all P 〈 0. 01 ）. Conclusions High levels of glucose and insulin can inhibit the proliferation and induce apoptosis of EPCs in mice. Insulin cannot reverse the inhibitory effect of high level of glucose on EPCs in mice.