新的吡唑衍生物对血管内皮细胞和A549肺癌细胞凋亡的影响及其作用机制研究
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摘要
血管内皮细胞功能的失调不仅引发血管疾病,还会导致人体的其它多种疾病。血管内皮细胞凋亡与动脉粥样硬化和多种血管退行性疾病有直接的关系。因此,抑制血管内皮细胞凋亡对防治这些重大血管疾病具有重要意义。我们的先前研究表明,去除血清和FGF-2可以诱导人脐静脉血管内皮细胞(HUVECs)凋亡,本论文利用该实验模型,从化学遗传学角度出发,以新的吡唑衍生物为工具,筛选血管内皮细胞凋亡抑制剂并研究其发挥作用的分子机制。
研究方法:
1.血管内皮细胞的提取和培养,参考Jaffe等的方法[Jaffe et al,1973]
2.倒置相差显微镜观察细胞形态变化
3.MTT检测细胞存活率
4.吖啶橙染色结合激光扫描共聚焦显微镜观察,检测细胞核凝集
5.TUNEL染色检测DNA片段化和细胞凋亡率
6.免疫荧光化学检测血管内皮细胞膜整连蛋白β4,p53的水平
7.荧光探针DCHF检测ROS表达水平
研究结果:
1乙烷基3-(o-氯苯基)-5-甲基-1H-g比唑-羧酸酯(4a)(100μM),乙烷基5-甲基-3-(o-硝基苯基)-1H-吡唑-4-羧酸酯(4b)(100μM),均能显著抑制HUVECs存活;1-苯基-3-p-甲苯基-1H-吡唑-5-ol(4f)(25μM)显著提高HUVECs存活率。
2乙烷基3-(o-氯苯基)-5-甲基-1-苯基-1H-吡唑-羧酸酯(4d,MPD)(20,25μM)抑制去除血清和FGF-2诱导的血管内皮细胞凋亡,但是在50,100μM时促进凋亡。
3 MPD(25μM)可显著抑制由于去除血清和FGF-2而导致的膜整连蛋白β4和p53蛋白水平的升高。
4 MPD(25μM)可降低细胞内ROS水平。
5在有血清和FGF-2存在时,MPD(25,50,100μM)对HUVECs形态和存活率没有影响。
6当分别用100μM 4a和50,100μM 4d处理A549肺癌细胞时,其生长受到显著抑制(p＜0.01),并诱导其凋亡。4b,3-(o-硝基苯基)-1-苯基-1H-5-ol(4e)以及4f对A549肺癌细胞生长没有明显影响。
结论:
1在无血清和FGF-2的条件下,4a(100μM),4b(100μM),4d(100μM)均能显著抑制HUVECs存活。4d(25μM),4f(25μM)显著提高HUVECs存活率。
2低浓度的MPD(25μM)抑制去除血清和FGF-2诱导的HUVECs凋亡,而高浓度的MPD(100μM)促进其凋亡。
3 MPD(25μM)可能通过下调膜整连蛋白β4和p53以及细胞内ROS水平抑制去除血清和FGF-2诱导的凋亡。
4在有血清和FGF-2存在时,MPD对HUVECs形态和存活率没有影响。
5 4a(100μM),MPD(50,100μM)均可诱导A549细胞凋亡。
Effects of new pyrazole derivatives on apoptosis of vascular endothelial cells and A549 lung cancer cells and the corresponding mechanisms
Vascular endothelial cells(VEC)form the inner lining of all blood vessels and function to maintain vascular tone and anticoagulant properties of blood vessels.VEC apoptosis plays important roles in many diseases,including atherosclerosis and cancer. Previously,we found that deprivation of serum and FGF-2 could induce HUVEC apoptosis.In this study,in the light of chemical genetics,we used new pyrazole derivatives as tools to study the molecular mechanism of apoptosis in HUVECs and A549 lung cancer cells.
METHODS:
1.HUVECs were gained as described previously[Jaffe et al.,1973].
2.The morphological changes were observed under a phase contrast microscope.
3.The cell proliferation was measured by MTT assay.
4.DNA nuclear fragmentation was analyzed by acridine orange staining.
5.The TdT-mediated dUTP nick end labeling technique was used to detect in situ nuclear DNA fragmentation and count apoptosis ratio.
6.The expressions and distributions of integrinβ4 and p53 were analysed by immunofluorescence assay.
7.The fluorescence probe,DCHF was used to analyze ROS level.
RESULTS:
1 When we treated HUVECs with Ethyl 3-(o-chlorophenyl)-5-methyl-1H-pyrazole-4-carboxylate(4a),Ethyl
5-methyl-3-(o-nitrophenyl)-1H-pyrazole-4-carboxylate(4b)at 100μM, respectively,the viability of HUVECs decreased obviously.But,when we treated HUVECs with 1-Phenyl-3-p-tolyl-1H-pyrazol-5-ol(4f)at 25μM,the viability of HUVECs increased obviously.
2 Ethyl 3-(o-chlorophenyl)-5-methyl-1-phenyl-1H-pyrazole-4-carboxylate(4d, MPD)(25μM)inhibited the apoptosis induced by deprivation of serum and FGF-2,but promoted the apoptosis at 100μM.
3 The high levels of integrinβ4 and p53 induced by the deprivation of serum and FGF-2 could be depressed by the treatment of MPD(25μM).
4 MPD(25μM)could depress the accumulation of intracellular ROS.
5 MPD(25,50,100μM)could not affect the morphology and viability of HUVECs in the present of serum and FGF-2.
6 When treated with 4a(100μM)and 4d(MPD)(50,100μM),respectively,A549 cell growth was obviously suppressed(p＜0.01)and the cells were induced to apoptosis.
7 4b,3-(o-nitrophenyl)-1-phenyl-1H-pyrazol-5-ol(4e),4f,had no obvious effects on A549 cell growth.
CONCLUSION:
1 In the absence of serum and FGF-2,4f(25μM)could increase the viability of HUVECs obviously,but 4a and 4b could depress the viability of HUVECs obviously at 100μM,respectively..
2 MPD inhibited the apoptosis of HUVECs induced by the deprivation of serum and FGF-2 at low concentration(25μM),but promoted apoptosis at high concentrations(50,100μM).
3 MPD(25μM)inhibited the apoptosis through down-regulating the levels of integrinβ4,p53,and ROS.
4 MPD(25,50,100μM)could not affect the viability of HUVECs in the present of serum and FGF-2.
5 The growth of A549 cells decreased after treatment with 4a at 100μM for 24 h or 48 h,respectively.
6 The growth of A549 cells decreased after treatment with MPD 50,100μM, respectively.
研究方法:
1.血管内皮细胞的提取和培养,参考Jaffe等的方法[Jaffe et al,1973]
2.倒置相差显微镜观察细胞形态变化
3.MTT检测细胞存活率
4.吖啶橙染色结合激光扫描共聚焦显微镜观察,检测细胞核凝集
5.TUNEL染色检测DNA片段化和细胞凋亡率
6.免疫荧光化学检测血管内皮细胞膜整连蛋白β4,p53的水平
7.荧光探针DCHF检测ROS表达水平
研究结果:
1乙烷基3-(o-氯苯基)-5-甲基-1H-g比唑-羧酸酯(4a)(100μM),乙烷基5-甲基-3-(o-硝基苯基)-1H-吡唑-4-羧酸酯(4b)(100μM),均能显著抑制HUVECs存活;1-苯基-3-p-甲苯基-1H-吡唑-5-ol(4f)(25μM)显著提高HUVECs存活率。
2乙烷基3-(o-氯苯基)-5-甲基-1-苯基-1H-吡唑-羧酸酯(4d,MPD)(20,25μM)抑制去除血清和FGF-2诱导的血管内皮细胞凋亡,但是在50,100μM时促进凋亡。
3 MPD(25μM)可显著抑制由于去除血清和FGF-2而导致的膜整连蛋白β4和p53蛋白水平的升高。
4 MPD(25μM)可降低细胞内ROS水平。
5在有血清和FGF-2存在时,MPD(25,50,100μM)对HUVECs形态和存活率没有影响。
6当分别用100μM 4a和50,100μM 4d处理A549肺癌细胞时,其生长受到显著抑制(p＜0.01),并诱导其凋亡。4b,3-(o-硝基苯基)-1-苯基-1H-5-ol(4e)以及4f对A549肺癌细胞生长没有明显影响。
结论:
1在无血清和FGF-2的条件下,4a(100μM),4b(100μM),4d(100μM)均能显著抑制HUVECs存活。4d(25μM),4f(25μM)显著提高HUVECs存活率。
2低浓度的MPD(25μM)抑制去除血清和FGF-2诱导的HUVECs凋亡,而高浓度的MPD(100μM)促进其凋亡。
3 MPD(25μM)可能通过下调膜整连蛋白β4和p53以及细胞内ROS水平抑制去除血清和FGF-2诱导的凋亡。
4在有血清和FGF-2存在时,MPD对HUVECs形态和存活率没有影响。
5 4a(100μM),MPD(50,100μM)均可诱导A549细胞凋亡。
Effects of new pyrazole derivatives on apoptosis of vascular endothelial cells and A549 lung cancer cells and the corresponding mechanisms
Vascular endothelial cells(VEC)form the inner lining of all blood vessels and function to maintain vascular tone and anticoagulant properties of blood vessels.VEC apoptosis plays important roles in many diseases,including atherosclerosis and cancer. Previously,we found that deprivation of serum and FGF-2 could induce HUVEC apoptosis.In this study,in the light of chemical genetics,we used new pyrazole derivatives as tools to study the molecular mechanism of apoptosis in HUVECs and A549 lung cancer cells.
METHODS:
1.HUVECs were gained as described previously[Jaffe et al.,1973].
2.The morphological changes were observed under a phase contrast microscope.
3.The cell proliferation was measured by MTT assay.
4.DNA nuclear fragmentation was analyzed by acridine orange staining.
5.The TdT-mediated dUTP nick end labeling technique was used to detect in situ nuclear DNA fragmentation and count apoptosis ratio.
6.The expressions and distributions of integrinβ4 and p53 were analysed by immunofluorescence assay.
7.The fluorescence probe,DCHF was used to analyze ROS level.
RESULTS:
1 When we treated HUVECs with Ethyl 3-(o-chlorophenyl)-5-methyl-1H-pyrazole-4-carboxylate(4a),Ethyl
5-methyl-3-(o-nitrophenyl)-1H-pyrazole-4-carboxylate(4b)at 100μM, respectively,the viability of HUVECs decreased obviously.But,when we treated HUVECs with 1-Phenyl-3-p-tolyl-1H-pyrazol-5-ol(4f)at 25μM,the viability of HUVECs increased obviously.
2 Ethyl 3-(o-chlorophenyl)-5-methyl-1-phenyl-1H-pyrazole-4-carboxylate(4d, MPD)(25μM)inhibited the apoptosis induced by deprivation of serum and FGF-2,but promoted the apoptosis at 100μM.
3 The high levels of integrinβ4 and p53 induced by the deprivation of serum and FGF-2 could be depressed by the treatment of MPD(25μM).
4 MPD(25μM)could depress the accumulation of intracellular ROS.
5 MPD(25,50,100μM)could not affect the morphology and viability of HUVECs in the present of serum and FGF-2.
6 When treated with 4a(100μM)and 4d(MPD)(50,100μM),respectively,A549 cell growth was obviously suppressed(p＜0.01)and the cells were induced to apoptosis.
7 4b,3-(o-nitrophenyl)-1-phenyl-1H-pyrazol-5-ol(4e),4f,had no obvious effects on A549 cell growth.
CONCLUSION:
1 In the absence of serum and FGF-2,4f(25μM)could increase the viability of HUVECs obviously,but 4a and 4b could depress the viability of HUVECs obviously at 100μM,respectively..
2 MPD inhibited the apoptosis of HUVECs induced by the deprivation of serum and FGF-2 at low concentration(25μM),but promoted apoptosis at high concentrations(50,100μM).
3 MPD(25μM)inhibited the apoptosis through down-regulating the levels of integrinβ4,p53,and ROS.
4 MPD(25,50,100μM)could not affect the viability of HUVECs in the present of serum and FGF-2.
5 The growth of A549 cells decreased after treatment with 4a at 100μM for 24 h or 48 h,respectively.
6 The growth of A549 cells decreased after treatment with MPD 50,100μM, respectively.
引文
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