葡萄糖对不同氧浓度下内皮祖细胞表达低氧诱导因子-1α的影响

Effects of glucose on the expression of hypoxia-inducible factor-1α of endothelial progenitor cells in different oxygen concentrations

目的 观察葡萄糖对低氧（1%氧浓度）和常氧（21%氧浓度）状态下内皮祖细胞表达低氧诱导因子-1α（HIF-1α）和血管内皮生长因子（VEGF）的影响,探讨低氧在糖尿病周围血管病发生中的可能作用.方法 常规培养健康人外周血来源的内皮祖细胞,加入不同浓度葡萄糖（5、10、33 mmol/L）分为A、B、C 3组,并在常氧和低氧条件下分别进行培养.实时荧光定量逆转录-聚合酶链反应（RT-PCR）法检测内皮祖细胞表达HIF-1α及VEGF的水平,免疫印迹法检测HIF-1α蛋白表达,ELISA法检测VEGF分泌水平.结果 （1）低氧组表达HIF-1α mRNA（A、B和C组分别为1.25±0.34、1.35±0.26和0.75±0.22）高于常氧组（A、B和C组分别为1.03 ±0.25、1.21±0.28和0.61±0.17）,差异具有统计学意义（A、B和C组t值分别为1.96,2.11,1.89,均P＜0.05）;而在相同氧浓度下,B组的内皮祖细胞表达HIF-1α mRNA高于A组和C组,差异具有统计学意义（低氧组F=23.54,P＜0.01;常氧组F=29.46,P＜0.01）.（2）在相同葡萄糖浓度下,低氧组和常氧组VEGF的蛋白表达差异无统计学意义（t值分别为0.933、1.258和1.001,均P＞0.05）.在低氧浓度下,VEGF的蛋白表达A、B和C组分别为2953±237、2473±205和1768±195,差异具有统计学意义（F=137.43,P=0.000）;在常氧浓度下,A、B和C组VEGF蛋白分别为2868±247、2377±197和1844±203,F=97.96,P=0.000.结论 高糖对低氧条件下的内皮祖细胞具有毒性作用,能减弱其表达HIF-1α和VEGF,可能在糖尿病周围血管病的发生中发挥一定作用.

Objective To observe the effects of various concentrations of glucose on hypoxiainducible factor-1α （HIF-1α） and vascular endothelial growth factor （VEGF） expressed by endothelial progenitor cells （EPCs）,and investigate the possible mechanism of hypoxia in the generation and development of diabetic peripheral vascular disease. Methods Different concentrations of glucose were added to the culture system of EPCs collected from the blood of healthy volunteers. According to the concentrations of glucose,the EPCs were divided into three groups Group A （[Glu]=5 mmol/L）,B （ [Glu] = 10 mmol/L） and C （[Glu]=33 mmol/L）,which were cultured separately in the normal （21%oxygen） and low oxygen environment （1% oxygen） in each group.The level of mRNA of HIF-1α and VEGF expressed by EPCs was detected by RT-PCR assays,and the expression of HIF-1α protein was measured by Western blot,and the level of VEGF secretion was measured by ELISA.ANOVA analysis was used for data statistics.Results （1）The HIF-1α mRNA expression in hypoxic group （ A = 1.25 ±0.34,B = 1.35 ±0.26,C =0.75 ± 0.22,respectively） was enhanced significantly in normoxic group（A=1.03±0.25,B=1.21±0.28,C =0.61 ±0.17,respectively）,（group A： t=1.96,P=0.03; group B：t=2.11 ;P = 0.03; group C： t = 1.89,P = 0.04 respectively）.Under the same oxygen concentrations,the mRNA expression level of HIF-1α in group B was significantly higher than those in group A and group C （ hypoxic group F = 23.54,P=0.000; normoxic group F=29.46,P=0.000）. （ 2 ） The protein expression levels of VEGF in hypoxic group and normoxic group were no statistical significance （ t = 0.933,1.258,1.001,respectively and all P〉0.05 ）under the same glucose concentrations.Under hypoxia condition,the protein expression levels of VEGF among group A,group B and group C（A =2953±237,B=2473±205,C=1768±195,respectively）were statistical significance （ F = 137.43 ,P =0.000）.The protein expression level of VEGF among group A,B and C was 2868 ± 247,2377 ± 197,1844 ± 203,respectively under normoxic condition （F=97.96,P=0.000）.Conclusion High level of glucose has toxicity to EPCs in the hypoxia environment,and attenuates the expression of HIF-1α and VEGF of EPCs,which might play an important role in the pathogenesis of diabetic peripheral vascular disease.