胰岛素对内皮祖细胞增殖、衰老及NO分泌的影响

Effects of insulin on proliferation, sene-scence and NO secretion of endothelial progenitor cells

目的:探讨胰岛素对内皮祖细胞(EPC)增殖能力、衰老程度以及分泌一氧化氮(NO)能力的影响.方法:采用密度梯度离心法获取大鼠骨髓单核细胞,在M199培养液中培养扩增EPC并进行鉴定.细胞分为对照组(常规M199培养液)和3个胰岛素干预组(在常规M199培养液中分别加入0.1,1,10nmol/L胰岛素),干预24h后硝酸酶还原法观察EPC分泌NO量的变化.干预7d后采用噻唑蓝(MTT)比色法观察胰岛素对EPC增殖能力的影响.衰老相关β-半乳糖苷酶(SA-β-Gal)染色观察EPC衰老率的变化.结果:在体外培养条件下,0.1,1nmol/L胰岛素组与对照组相比EPC数量显著增加[A570nm:(0.262±0.015),(0.290±0.021)vs对照组(0.232±0.021),P均<0.01];衰老率显著降低[(17.1±1.6)%,(11.7±1.8)%vs对照组(27.9±13.5)%,P均<0.05];培养上清中NO浓度显著提高[μmol/L,(42.4±1.4),(54.4±5.8)vs对照组(35.7±1.3),P均<0.05],而10nmol/L胰岛素组的各项指标与对照组差别不明显.结论:低浓度胰岛素能促进EPC的增殖,抑制EPC衰老,促进其分泌NO.

AIM： To investigate the effects of insulin on endothelial progenitor cell （EPC） proliferation, senescence and nitric oxide （NO） secretion. METHODS： Mononuelear cells were collected from rat bone marrow by density gradient centrifugation, cultured with Medium 199, and were identified to be EPC on day 7 by ilk-1 and AC133 double staining. EPC were harvested and incubated with vehicle or insulin （0.1, 1, 10 nmol/L）. After 24 h of incubation, NO secretion of EPC was assessed by Griess reaction. After 7 d of incubation, proliferative capacity was measured by MTT assay, and senescent level was measured through senescence associated β-galactosidase （SA-β-Gal） staining. RESULTS： Compared with control group, 0. 1 and 1 nmol/L insulin group showed higher EPC proliferative capacity （absorbance ： 0.262 ±0.015,0.290 ±0.021 vs control 0.232 ±0.021, P〈0.01） , lower EPC senescence [（17.1 ±1.6）% , （11.7 ± 1.8）% vs control （27.9 ±13.5）%, P〈0.05] after 7 d of incubation, and higher NO production [ umol/L, （42.4 ± 1.4 ）, （ 54.4 ± 5.8 ） vs control （ 35.7 ± 1.3 ）, P 〈 0.05 ] after 24 h incubation, while 10 nmol/L insulin group showed no or only minor difference as compared with control group. CONCLUSION： Low-level insulin can stimulate EPC proliferation, inhibit its senescence, and enhance its NO secretion capacity.