中山大学学报（医学科学版）
中山大學學報（醫學科學版）
중산대학학보（의학과학판）
JOURNAL OF SUN YAT-SEN UNIVERSITY(MEDICAL SCIENCES)
2004年
6期
496-503
,共8页
区景松%欧志君%Kirkwood A.Pritchard Jr
區景鬆%歐誌君%Kirkwood A.Pritchard Jr
구경송%구지군%Kirkwood A.Pritchard Jr
热休克蛋白 90%一氧化氮合酶%一氧化氮%氧自由基%内皮细胞
熱休剋蛋白 90%一氧化氮閤酶%一氧化氮%氧自由基%內皮細胞
열휴극단백 90%일양화담합매%일양화담%양자유기%내피세포
heat shock protein 90%nitric oxide synthase%nitric oxide%superoxide%endothelial cell
[目的] Trichloroethylene (TRI) 是一种与先天性心脏病发生有关的环境污染物,我们最近报道在分裂期的内皮细胞( EC)中, TRI可以通过减少热休克蛋白 90( hsp90)与内皮一氧化氮合酶( eNOS)结合,从而抑制刺激状态下一氧化氮( NO)产生和增加刺激状态下 eNOS依赖的超氧阴离子自由基(O2)产生和抑制血管内皮生长因子刺激的内皮细胞分裂.但 TRI在融合期 EC中的影响仍未清楚.本实验目的是探讨 TRI对融合期 EC的影响.[方法] TRI 5 μ mol/L 预处理融合期 EC,用钙离子导体 A23187 5 μ mol/L 刺激,然后,检测 NO与(O2)的产生以了解 EC和 eNOS的功能情况.用免疫印迹方法检查 eNOS,位点于丝氨酸 1179的磷酸化 eNOS,与 eNOS结合的 hsp90水平来决定 eNOS的激活状态.[结果] TRI减少 A23187刺激状态下亚硝酸盐和硝酸盐的产生从 (1.16 ± 0.15) nmol· mg- 1到 (0.91 ± 0.3) nmol· mg- 1 (P < 0.05);在对照情况下, L- NAME 增加 A23187 刺激状态下的(O2)产生从 (0.015 ± 0.007) nmol/(min· mg)到 (0.044 ± 0.008) nmol/(min · mg) (P< 0.05);在 TRI处理的培养中, L- NAME 减少 A23187刺激状态下的产生从 (0.057 ± 0.022) nmol/(min · mg)到 (0.039 ± 0.005) nmol/(min · mg) (P< 0.05),并且 oxypurinol减少 A23187刺激状态下的(O2)产生从 (0.057 ± 0.022) nmol/(min · mg)到 (0.034 ± 0.012) nmol/(min · mg) (P< 0.05); TRI 对 eNOS, p- eNOS 和 hsp90的表达无影响,但减少 hsp90与 eNOS的结合.[结论] 这些数据表明,在融合期 EC中, TRI 不但改变 hsp90与 eNOS 结合,使 eNOS 从产生 NO变为产生 (O2),而且激活第二氧化酶-黄嘌呤氧化酶,而增加(O2)产生.这些内皮功能的改变可能导致许多心血管疾病的发生.
[目的] Trichloroethylene (TRI) 是一種與先天性心髒病髮生有關的環境汙染物,我們最近報道在分裂期的內皮細胞( EC)中, TRI可以通過減少熱休剋蛋白 90( hsp90)與內皮一氧化氮閤酶( eNOS)結閤,從而抑製刺激狀態下一氧化氮( NO)產生和增加刺激狀態下 eNOS依賴的超氧陰離子自由基(O2)產生和抑製血管內皮生長因子刺激的內皮細胞分裂.但 TRI在融閤期 EC中的影響仍未清楚.本實驗目的是探討 TRI對融閤期 EC的影響.[方法] TRI 5 μ mol/L 預處理融閤期 EC,用鈣離子導體 A23187 5 μ mol/L 刺激,然後,檢測 NO與(O2)的產生以瞭解 EC和 eNOS的功能情況.用免疫印跡方法檢查 eNOS,位點于絲氨痠 1179的燐痠化 eNOS,與 eNOS結閤的 hsp90水平來決定 eNOS的激活狀態.[結果] TRI減少 A23187刺激狀態下亞硝痠鹽和硝痠鹽的產生從 (1.16 ± 0.15) nmol· mg- 1到 (0.91 ± 0.3) nmol· mg- 1 (P < 0.05);在對照情況下, L- NAME 增加 A23187 刺激狀態下的(O2)產生從 (0.015 ± 0.007) nmol/(min· mg)到 (0.044 ± 0.008) nmol/(min · mg) (P< 0.05);在 TRI處理的培養中, L- NAME 減少 A23187刺激狀態下的產生從 (0.057 ± 0.022) nmol/(min · mg)到 (0.039 ± 0.005) nmol/(min · mg) (P< 0.05),併且 oxypurinol減少 A23187刺激狀態下的(O2)產生從 (0.057 ± 0.022) nmol/(min · mg)到 (0.034 ± 0.012) nmol/(min · mg) (P< 0.05); TRI 對 eNOS, p- eNOS 和 hsp90的錶達無影響,但減少 hsp90與 eNOS的結閤.[結論] 這些數據錶明,在融閤期 EC中, TRI 不但改變 hsp90與 eNOS 結閤,使 eNOS 從產生 NO變為產生 (O2),而且激活第二氧化酶-黃嘌呤氧化酶,而增加(O2)產生.這些內皮功能的改變可能導緻許多心血管疾病的髮生.
[목적] Trichloroethylene (TRI) 시일충여선천성심장병발생유관적배경오염물,아문최근보도재분렬기적내피세포( EC)중, TRI가이통과감소열휴극단백 90( hsp90)여내피일양화담합매( eNOS)결합,종이억제자격상태하일양화담( NO)산생화증가자격상태하 eNOS의뢰적초양음리자자유기(O2)산생화억제혈관내피생장인자자격적내피세포분렬.단 TRI재융합기 EC중적영향잉미청초.본실험목적시탐토 TRI대융합기 EC적영향.[방법] TRI 5 μ mol/L 예처리융합기 EC,용개리자도체 A23187 5 μ mol/L 자격,연후,검측 NO여(O2)적산생이료해 EC화 eNOS적공능정황.용면역인적방법검사 eNOS,위점우사안산 1179적린산화 eNOS,여 eNOS결합적 hsp90수평래결정 eNOS적격활상태.[결과] TRI감소 A23187자격상태하아초산염화초산염적산생종 (1.16 ± 0.15) nmol· mg- 1도 (0.91 ± 0.3) nmol· mg- 1 (P < 0.05);재대조정황하, L- NAME 증가 A23187 자격상태하적(O2)산생종 (0.015 ± 0.007) nmol/(min· mg)도 (0.044 ± 0.008) nmol/(min · mg) (P< 0.05);재 TRI처리적배양중, L- NAME 감소 A23187자격상태하적산생종 (0.057 ± 0.022) nmol/(min · mg)도 (0.039 ± 0.005) nmol/(min · mg) (P< 0.05),병차 oxypurinol감소 A23187자격상태하적(O2)산생종 (0.057 ± 0.022) nmol/(min · mg)도 (0.034 ± 0.012) nmol/(min · mg) (P< 0.05); TRI 대 eNOS, p- eNOS 화 hsp90적표체무영향,단감소 hsp90여 eNOS적결합.[결론] 저사수거표명,재융합기 EC중, TRI 불단개변 hsp90여 eNOS 결합,사 eNOS 종산생 NO변위산생 (O2),이차격활제이양화매-황표령양화매,이증가(O2)산생.저사내피공능적개변가능도치허다심혈관질병적발생.
[ Objective] Trichloroetheylene (TRI) is an environmental pollutant that has been linked to congenital heart defects. Our earlier report showed that TRI inhibited stimulated nitric oxide (NO) production and increased stimulated endothelial nitric oxide synthase (eNOS)- dependent superoxide anion (O2) production by decreasing heat shock protein 90(hsp90) associated with eNOS and limited vascular endothelial growth factor- stimulated proliferation on proliferating endothelial cells (EC). The effects of TRI on confluent EC remained unknown. The aim of this study was to test the effects of TRI on confluent EC. [ Method] Confluent EC were pretreated with TRI (5 μ mol/L) and then stimulated with the calcium ionophore, A23187 (5 μ mol/L), to determine changes in EC and eNOS functions with respect to NO and (O2) generation. Immunoblots of eNOS, phosphorylation of eNOS (P- eNOS) at serine 1179 (S1179) and the levels of associated hsp90 with eNOS were used to define the activation state of eNOS. [ Results] TRI decreased A23187- stimulated nitrite + nitrate production from ( 1.16± 0.15) nmol· mg- 1 to ( 0.91± 0.3) nmol· mg- 1 (P < 0.05). In controls, L- nitroargininemethylester (L- NAME) increased A23187- stimulated production from ( 0.015± 0.007) nmol /( min· mg) to ( 0.044± 0.008) nmol /( min· mg) (P < 0.05). In TRI- treated cultures, however, L- NAME decreased A23187- stimulated (O2) production from ( 0.057± 0.022) nmol /( min· mg) to ( 0.039± 0.005) nmol /( min· mg) (P < 0.05) and oxypurinol decreased A23187- stimulated (O2) production from(0 05± 0 022)nmol/( min· mg) to 0.034± 0.012 nmol/( min· mg) (P < 0.05). TRI has no effect on eNOS, p- eNOS,and hsp90 expression but decreased hsp90 association with eNOS. [ Conclusion] These data show that TRI not only alters hsp90 interaction with eNOS and shifts eNOS from · NO to (O2) generation, but also activates a second oxidative enzyme, xanthine oxidase, to increase (O2) generation in confluent EC. Such changes in endothelial functions may contribute to many cardiovascular diseases.
