过氧化氢诱导人血管内皮细胞凋亡及HSP70和Bcl-2基因蛋白表达的研究

Induction of apoptosis and expression of heat shock protein 70 and Bcl-2 genes in human vascular endothelial cells

目的 :探讨过氧化氢 (H2 O2 )对血管内皮细胞 (VEC)损伤机制和细胞凋亡的调控。方法 :通过人脐静脉内皮细胞株 (ECV 30 4)体外培养 ,采用流式细胞技术、原位末端脱氧核苷酸转移酶标记法和分子生物学分析相结合的方法观察 H2 O2 对 VEC中热休克蛋白 70 (HSP70 )和 Bcl 2基因蛋白表达及细胞凋亡的影响。结果 :两种检测方法细胞凋亡率变化趋势一致 :10 0、30 0和 5 0 0 mm ol/ L H2 O2 刺激后 ,VEC中 HSP70蛋白表达由(12 .15± 3.0 3) %分别增至 (2 3.80± 8.74) %、(35 .35± 6 .6 8) %和 (35 .85± 5 .83) % ,差异显著 (P<0 .0 5或 P<0 .0 1) ;而 Bcl 2表达下调 ,由 (2 4.90± 2 .95 ) %分别下降至 (19.35± 3.36 ) %、(18.75± 3.33) %和 (13.0 5±2 .93) % (P<0 .0 5或 P<0 .0 1)。 H2 O2 可诱导 VEC发生凋亡 ,并在一定范围内呈量效关系。结论 :细胞凋亡是血管内皮细胞受损的主要方式之一 ,HSP70与 Bcl 2表达改变可能共同参与了急性呼吸窘迫综合征的发病。

Objective:To investigate the effect of hydrogen peroxide (H 2O 2) on apoptosis induction and its regulation mechanism in human vascular endothelial cells (VECs).Methods:VECs were cultured in vitro.Flow cytometry,terminal deoxynucleotidyl transferasemediated dUTP nick endlabeling (TUNEL) and molecular biology method were employed to observe occurrence of apoptosis,and the gene as well as protein expression of heat shock protein 70 (HSP70) and Bcl2.Results:Similar results were obtained by two different methods for apoptosis determination.Treatment with H 2O 2 at doses of 100,300,and 500 mmol/L,respectively,resulted in upregulation of HSP70 from (12 15±3 03)% to (23 80±8 74)%,(35 35±6 68)%,and (35 85±5 83)% ( P <0 05 or P <0 01),with downregulation of Bcl2 from (24 90±2 95)% to (19 35±3 36)%,(18 75±3 33)%,(13 05±2 93)% ( P <0 05 or P <0 01),respectively.H 2O 2 was able to induce apoptosis in human VECs,showing dosedependent response within certain extent.Conclusions:Apoptosis appears to be one of important mechanisms underlying the damage of human VECs.Apoptosis and changes in HSP70,Bcl2 gene/protein expression in human VECs might be involved in the pathogenesis of acute respiratory distress syndrome.