中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
1期
38-42
,共5页
包永琴%马景学%王淑芬%吕兰存%杜颖华
包永琴%馬景學%王淑芬%呂蘭存%杜穎華
포영금%마경학%왕숙분%려란존%두영화
血管内皮生长因子%二十碳五烯酸%血管生成%细胞周期
血管內皮生長因子%二十碳五烯痠%血管生成%細胞週期
혈관내피생장인자%이십탄오희산%혈관생성%세포주기
Vascular endothelial growth factor%Eicosapentaenoic acid%Angiogenesis%Cellular cycle
背景 研究表明二十碳五烯酸(EPA)作为主要的脂质参与人体的生物代谢活动,抑制血管内皮生长因子(VEGF)的产生、阻滞血管平滑肌细胞的内移和增生.而眼部多种疾病与新生血管的形成有关.目的 探讨EPA对VEGF诱导的人脐静脉内皮细胞(UVEC)增生的抑制作用.方法 对人UVEC株培养和传代,并用Ⅷ因子相关抗原多克隆抗体进行免疫细胞化学鉴定.应用免疫组织化学法检测EPA对VEGF受体2(Flk-1)在人UVEC中表达的影响;用MTT比色法检测不同质量浓度EPA作用不同时间后对非VEGF诱导及VEGF诱导的人UVEC增生的抑制率;用流式细胞术检测EPA对VEGF诱导的人UVEC细胞周期的影响.结果 培养和传代的人UVEC呈纺锤形及鹅卵石样排列,Ⅷ因子相关抗原鉴定呈阳性反应.EPA作用后,Flk-1在人UVEC中的阳性染色强度明显弱于对照组,Flk-1阳性细胞数明显减少.6个质量浓度组的EPA对VEGF诱导及非VEGF诱导的人UVEC的增生均有明显的抑制作用,其作用呈质量浓度依赖性(F=23.072,P=0.000);各质量浓度组的EPA对VEGF诱导的人UVEC的抑制作用强于非VEGF诱导的人UVEC,差异有统计学意义(F=41.417,P=0.000).各质量浓度组EPA对VEGF诱导的人UVEC作用24、48、72 h后,其细胞数逐渐降低(F=87.823,P=0.000),但作用时间对其抑制作用无明显影响(F=1.495,P=0.236).EPA使VEGF诱导的人UVEC细胞阻滞在G0/G1期,EPA组G0/G1期细胞数比例为(75.83±1.56)%,对照组为(68.62±1.44)%,差异有统计学意义(t=-5.88,P=0.00);EPA组与对照组均未发现凋亡细胞.结论 EPA能够通过阻止人血管内皮细胞的DNA合成而明显抑制VEGF诱导的人UVEC的增生,其抑制作用呈剂量依赖性;EPA作用后人UVEC中Flk-1的表达下调,提示EPA可能有抗血管生成的作用.
揹景 研究錶明二十碳五烯痠(EPA)作為主要的脂質參與人體的生物代謝活動,抑製血管內皮生長因子(VEGF)的產生、阻滯血管平滑肌細胞的內移和增生.而眼部多種疾病與新生血管的形成有關.目的 探討EPA對VEGF誘導的人臍靜脈內皮細胞(UVEC)增生的抑製作用.方法 對人UVEC株培養和傳代,併用Ⅷ因子相關抗原多剋隆抗體進行免疫細胞化學鑒定.應用免疫組織化學法檢測EPA對VEGF受體2(Flk-1)在人UVEC中錶達的影響;用MTT比色法檢測不同質量濃度EPA作用不同時間後對非VEGF誘導及VEGF誘導的人UVEC增生的抑製率;用流式細胞術檢測EPA對VEGF誘導的人UVEC細胞週期的影響.結果 培養和傳代的人UVEC呈紡錘形及鵝卵石樣排列,Ⅷ因子相關抗原鑒定呈暘性反應.EPA作用後,Flk-1在人UVEC中的暘性染色彊度明顯弱于對照組,Flk-1暘性細胞數明顯減少.6箇質量濃度組的EPA對VEGF誘導及非VEGF誘導的人UVEC的增生均有明顯的抑製作用,其作用呈質量濃度依賴性(F=23.072,P=0.000);各質量濃度組的EPA對VEGF誘導的人UVEC的抑製作用彊于非VEGF誘導的人UVEC,差異有統計學意義(F=41.417,P=0.000).各質量濃度組EPA對VEGF誘導的人UVEC作用24、48、72 h後,其細胞數逐漸降低(F=87.823,P=0.000),但作用時間對其抑製作用無明顯影響(F=1.495,P=0.236).EPA使VEGF誘導的人UVEC細胞阻滯在G0/G1期,EPA組G0/G1期細胞數比例為(75.83±1.56)%,對照組為(68.62±1.44)%,差異有統計學意義(t=-5.88,P=0.00);EPA組與對照組均未髮現凋亡細胞.結論 EPA能夠通過阻止人血管內皮細胞的DNA閤成而明顯抑製VEGF誘導的人UVEC的增生,其抑製作用呈劑量依賴性;EPA作用後人UVEC中Flk-1的錶達下調,提示EPA可能有抗血管生成的作用.
배경 연구표명이십탄오희산(EPA)작위주요적지질삼여인체적생물대사활동,억제혈관내피생장인자(VEGF)적산생、조체혈관평활기세포적내이화증생.이안부다충질병여신생혈관적형성유관.목적 탐토EPA대VEGF유도적인제정맥내피세포(UVEC)증생적억제작용.방법 대인UVEC주배양화전대,병용Ⅷ인자상관항원다극륭항체진행면역세포화학감정.응용면역조직화학법검측EPA대VEGF수체2(Flk-1)재인UVEC중표체적영향;용MTT비색법검측불동질량농도EPA작용불동시간후대비VEGF유도급VEGF유도적인UVEC증생적억제솔;용류식세포술검측EPA대VEGF유도적인UVEC세포주기적영향.결과 배양화전대적인UVEC정방추형급아란석양배렬,Ⅷ인자상관항원감정정양성반응.EPA작용후,Flk-1재인UVEC중적양성염색강도명현약우대조조,Flk-1양성세포수명현감소.6개질량농도조적EPA대VEGF유도급비VEGF유도적인UVEC적증생균유명현적억제작용,기작용정질량농도의뢰성(F=23.072,P=0.000);각질량농도조적EPA대VEGF유도적인UVEC적억제작용강우비VEGF유도적인UVEC,차이유통계학의의(F=41.417,P=0.000).각질량농도조EPA대VEGF유도적인UVEC작용24、48、72 h후,기세포수축점강저(F=87.823,P=0.000),단작용시간대기억제작용무명현영향(F=1.495,P=0.236).EPA사VEGF유도적인UVEC세포조체재G0/G1기,EPA조G0/G1기세포수비례위(75.83±1.56)%,대조조위(68.62±1.44)%,차이유통계학의의(t=-5.88,P=0.00);EPA조여대조조균미발현조망세포.결론 EPA능구통과조지인혈관내피세포적DNA합성이명현억제VEGF유도적인UVEC적증생,기억제작용정제량의뢰성;EPA작용후인UVEC중Flk-1적표체하조,제시EPA가능유항혈관생성적작용.
Background Eicosapentaenoic acid(EPA)function as the critical lipid mediators involved in several biological events in human body and play important role in suppressing the genesis of vascular endothelial growth factor (VEGF),migration and proliferation of vascular endothelial cells.Many ocular diseases were proved to be associated with neovascularization.Objecfive The purpose of this study was to investigate the inhibitory effect of EPA on the proliferation of human umbilical vein endothelial cells (HUVEC) indueed by VEGF. Methods HUVEC strain was cultured and passaged,and difierent concentrations of EPA were added to the medium with and without VEGF.The cultured cells were identified by antiofactor Ⅷ polyclonal antibody.The suppressing role of different concentrations of EPA on the proliferation of VEGF-induced or-uninduced HUVEC was assessed by MTT method.The influence of difierent concentrations of EPA on the cellular cycle of VEGF-induced HUVEC was assayed using flow eytometry.The expression of Flk-1,a receptor of VEGF,in the HUVEC Was detected by immunohistochemistry. Results Cultured HUVEC showed the ftlsiform in shape and presented with the cobblestone-like arrangement with the positive response for Ⅷ factor-related antigen.Various concentrations of EPA showed obviously inhibitory effect on VEGF-induced or-unindueed HUVEC at a dose-dependent manner (F=23.072.P=0.000).The inhibitory ability of EPA on VEGF-induced HUVEC was stronger than VEGF-uninduced HUVEC(F=41.417,P=0.000).In 24,48 and 72 hours,the action of EPA on the proliferation of HUVEC was gradually enhanced with the prolong of time(F=1.495,P=0.236).Cell cycle analysis indicated that EPA arrested VEGF-induced HUVEC in G0/G1 phase.The ratio of HUVEC in G0/G1 phase in EPA group was(75.83±1.56)%,and that in control groups was(68.62±1.44)%,showing a significant difference between them(t=-5.88,P=0.00),and no apoptosis of HUVEC was found in both groups.Flk-1 was strongly expressed in the cellular nucleus and cytoplasm in control group.However,the positive expressing intensity of Flk-1 in the HUVEC weakened,and the positive cell number was evidently less in EPA group. Conclusion EPA can inhibit the proliferation of VEGF induced HUVEC through arresting the synthesis of DNA of HUVEC and downregulate the expression of Flk-1 in HUVEC.These results suggest that EPA might exert an antiangiogenic effect.
