Objective： To observe the effect of Xuefu Zhuyu Decoction （血府逐瘀汤）-containing serum （XFZYD-CS） on endothelial progenitor cell （EPC） tube formation in vitro. Methods： Mononuclear cells from rat bone marrow ...Objective： To observe the effect of Xuefu Zhuyu Decoction （血府逐瘀汤）-containing serum （XFZYD-CS） on endothelial progenitor cell （EPC） tube formation in vitro. Methods： Mononuclear cells from rat bone marrow were prepared in a Ficoll density gradient centrifuge. EPCs were separated by the differential attachment method, and observed with inverted microscope for the effect of XFZYD-CS on EPC tube formation. Results： After one day, EPCs exposed to the serum containing 5%, 10% and 15% XFZYD-CS formed typical tubes or vessel networks. The tube formation time was two days ahead of the control group and the size of most tubes in the serum groups was smaller than in the control group. Conclusion： XFZYD-CS could induce EPC angiogenesis and hasten tube formation, especially in capillary vessels. The study provides experimental evidence for the plausibility of Xuefu Zhuyu Decoction in the treatment of ischemic diseases.展开更多
目的:为探讨桂皮醛对糖尿病视网膜病变新生血管的作用及机制,观察了桂皮醛对血管内皮生长因子(VEGF)诱导EA. hy926细胞增殖、迁移、成管以及Janus激酶2/信号传导与转录激活因子3(JAK2/STAT3)通路的影响。方法:将EA.hy926细胞分成空白组...目的:为探讨桂皮醛对糖尿病视网膜病变新生血管的作用及机制,观察了桂皮醛对血管内皮生长因子(VEGF)诱导EA. hy926细胞增殖、迁移、成管以及Janus激酶2/信号传导与转录激活因子3(JAK2/STAT3)通路的影响。方法:将EA.hy926细胞分成空白组、模型组(7μg·L-1VEGF),VEGF+桂皮醛(60,90,120,150μmol·L-1)组,分别采用噻唑蓝(MTT)比色法和划痕实验检测桂皮醛对VEGF诱导EA. hy 926细胞增殖和迁移作用的影响;将EA. hy 926细胞分成空白组,模型组(7μg·L-1VEGF),VEGF+桂皮醛(90,150μmol·L-1)组,采用管腔形成实验检测桂皮醛对VEGF诱导EA. hy 926细胞成管作用的影响;将EA. hy 926细胞分成空白组,模型组(7μg·L-1VEGF),VEGF+AG490 (50μmol·L-1)组,VEGF+桂皮醛(90μmol·L-1)组,VEGF+桂皮醛(150μmol·L-1)组,VEGF+桂皮醛(150μmol·L-1)+AG490(50μmol·L-1)组,采用蛋白免疫印迹法(Western blot)检测桂皮醛对VEGF诱导EA. hy 926细胞JAK2/STAT3通路的影响。结果:与空白组比较,模型组能够显著地促进EA. hy 926细胞增殖和迁移(P <0. 01)。与模型组比较,桂皮醛(60,90,120,150μmol·L-1)组能显著抑制VEGF诱导EA. hy 926细胞的增殖和迁移(P <0. 01)。与空白组比较,VEGF对EA. hy 926细胞成管具有一定的促进作用,成管的节点数、交叉点数、网眼数和血管分支数均有增加,但无统计学差异。与模型组比较,桂皮醛(90,150μmol·L-1)组对成管的节点数、交叉点数和网眼数均有明显抑制作用(P <0. 05,P <0. 01)。与空白组比较,模型组p-JAK2,p-STAT3,STAT3蛋白表达明显升高(P <0. 05,P <0. 01)。与模型组比较,桂皮醛(150μmol·L-1)能够显著抑制VEGF引起的p-JAK2,p-STAT3,STAT3蛋白表达升高(P <0. 01),桂皮醛(90μmol·L-1)能够显著抑制VEGF引起的p-STAT3,STAT3蛋白表达升高(P <0. 05,P <0. 01)。结论:桂皮醛对VEGF诱导EA. hy 926细胞的增殖、迁移、成管具有明显的抑制作用,该作用与抑制JAK2/STAT3通路的激活有关。展开更多
AIMTo determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs).ME...AIMTo determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs).METHODShRVECs transfected with peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) siRNA were incubated for 24h and then placed into a normoxic (20%, O<sub>2</sub>) or hypoxic (1%, O<sub>2</sub>) environment for another 16h. PGC-1α mRNA and protein levels were detected by real-time PCR and Western blot. VEGF mRNA and protein levels were detected by real-time PCR and ELISA. Cell proliferation was evaluated by BrdU incorporation assay. Forty-eight hours after siRNA transfection, hRVECs were planted into Matrigel-coated plates and cultured under normoxic (20%, O<sub>2</sub>) or hypoxic (1%, O<sub>2</sub>) conditions for another 48h. The tube formation of hRVECs was observed under an optical microscope and quantified by counting the number of branch points and calculating the total tube length.RESULTSPGC-1α mRNA and protein levels were significantly reduced by PGC-1α siRNA, and VEGF mRNA and protein levels also decreased significantly. The percentage of BrdU-labeled cells in siPGC-1α groups were significantly decreased compared with control siRNA groups under normoxia and hypoxia in cell proliferation assay. In the tube formation assay, PGC-1α siRNA treated cells formed significantly fewer tubes.CONCLUSIONBlocking PGC-1α expression can inhibit VEGF expression in hRVECs and inhibit their ability to form tubes under both normoxic and hypoxic conditions.展开更多
目的探讨映山红花总黄酮(total flavones of rhododendra,TFR)促大鼠脑血管内皮细胞体外形成血管作用及与VEGFR_(2)和神经源性硫化氢(H_(2)S)的关系。方法采用大鼠脑血管内皮细胞单独培养及和与海马神经元共培养,分别采用不同的实验方...目的探讨映山红花总黄酮(total flavones of rhododendra,TFR)促大鼠脑血管内皮细胞体外形成血管作用及与VEGFR_(2)和神经源性硫化氢(H_(2)S)的关系。方法采用大鼠脑血管内皮细胞单独培养及和与海马神经元共培养,分别采用不同的实验方法检测细胞增殖、迁移、成管及H_(2)S含量和钙离子荧光强度,包括CCK-8法、细胞划痕法、Transwell法、基质胶成管、H_(2)S试剂盒及钙离子荧光探针法。结果在单独培养的大鼠脑血管内皮细胞上,H_(2)S供体NaHS(200μmol·L^(-1))和TFR(90、270、810 mg·L^(-1))对大鼠脑血管内皮细胞的增殖、迁移、成管及[Ca^(2+)]i荧光强度都有明显的促进作用。而VEGFR_(2)阻断剂SU5416(10μmol·L^(-1))可抑制TFR的促进内皮细胞增殖、迁移和形成血管及[Ca^(2+)]i荧光强度;在与海马神经元共培养的大鼠脑血管内皮细胞上,TFR显著地升高共培养中H_(2)S含量,并被CBS抑制剂AOAA(200μmol·L^(-1))抑制。与此同时,TFR明显地促进共培养中大鼠脑血管内皮细胞的形成血管作用,并可被AOAA和VEGFR_(2)阻断剂SU5416显著地抑制。结论TFR在体外可通过VEGFR_(2)升高[Ca^(2+)]i来促进脑血管内皮细胞形成血管,并可通过诱导神经元中CBS生成H_(2)S作用于大鼠脑血管内皮细胞的VEGFR_(2)来促进血管形成。展开更多
目的:探讨微小核糖核酸(microRNA)-24(miR-24)对内皮型一氧化氮合酶(eNOS)基因表达调节的分子机制及其对血管内皮细胞增殖、迁移和管腔形成能力的影响。方法:构建miR-24及其反义序列的高表达质粒,分别转染人脐静脉内皮细胞(HUVECs),根...目的:探讨微小核糖核酸(microRNA)-24(miR-24)对内皮型一氧化氮合酶(eNOS)基因表达调节的分子机制及其对血管内皮细胞增殖、迁移和管腔形成能力的影响。方法:构建miR-24及其反义序列的高表达质粒,分别转染人脐静脉内皮细胞(HUVECs),根据转染质粒将实验细胞分为:miR-24高表达组、miR-24干扰组和空白质粒对照组。用四甲基偶氮唑盐(MTT)检测HUVECs增殖能力,划痕和Transwell试验检测细胞的迁移能力,人工基底膜检测细胞的管腔形成能力;分别用逆转录-聚合酶链反应(RT-PCR)、蛋白免疫印迹法(Western blotting)检测eNOS和Sp1转录因子m RNA和蛋白表达水平。结果:(1)与空白质粒对照组比较,miR-24高表达组细胞增殖能力降低45.45%(0.36±0.04 vs 0.66±0.08,P<0.05);miR-24高表达组细胞迁移速度明显减缓,且迁移数目降低74.75%(30.25±3.78 vs 119.80±10.94,P<0.01),未能形成明显管腔样结构。(2)与空白质粒对照组比,miR-24高表达组eNOS m RNA降低46.2%(0.49±0.02vs 0.91±0.01,P<0.05),蛋白表达减少49.07%(0.55±0.05 vs 1.08±0.05,P<0.05);同时Sp1 m RNA降低44.9%(0.49±0.01 vs 0.89±0.02,P<0.05),其蛋白质表达量也相应减少54.90%(0.46±0.02 vs 1.02±0.04,P<0.05)。在miR-24抑制组中,上述指标较空白质粒对照组降低,但比miR-24高表达组显著升高,特别是小管形成数量、及管腔长度与空白质粒对照组相近。结论:miR-24显著抑制HUVECs的增殖、迁移和管腔形成的能力,并且与miR-24对eNOS的表达调控有关;miR-24明显抑制eNOS表达,Sp1的参与可能是这一调节过程的重要分子机制之一。展开更多
基金Supported by the National Natural Science Foundation of China (No.30772877)Basic Research Fund of China Academy of Chinese Medical Sciences(No.ZZ2006039)Fujian Academy of Integrative Medicine Foundation(No.3000-905010805)
文摘Objective： To observe the effect of Xuefu Zhuyu Decoction （血府逐瘀汤）-containing serum （XFZYD-CS） on endothelial progenitor cell （EPC） tube formation in vitro. Methods： Mononuclear cells from rat bone marrow were prepared in a Ficoll density gradient centrifuge. EPCs were separated by the differential attachment method, and observed with inverted microscope for the effect of XFZYD-CS on EPC tube formation. Results： After one day, EPCs exposed to the serum containing 5%, 10% and 15% XFZYD-CS formed typical tubes or vessel networks. The tube formation time was two days ahead of the control group and the size of most tubes in the serum groups was smaller than in the control group. Conclusion： XFZYD-CS could induce EPC angiogenesis and hasten tube formation, especially in capillary vessels. The study provides experimental evidence for the plausibility of Xuefu Zhuyu Decoction in the treatment of ischemic diseases.
文摘目的:为探讨桂皮醛对糖尿病视网膜病变新生血管的作用及机制,观察了桂皮醛对血管内皮生长因子(VEGF)诱导EA. hy926细胞增殖、迁移、成管以及Janus激酶2/信号传导与转录激活因子3(JAK2/STAT3)通路的影响。方法:将EA.hy926细胞分成空白组、模型组(7μg·L-1VEGF),VEGF+桂皮醛(60,90,120,150μmol·L-1)组,分别采用噻唑蓝(MTT)比色法和划痕实验检测桂皮醛对VEGF诱导EA. hy 926细胞增殖和迁移作用的影响;将EA. hy 926细胞分成空白组,模型组(7μg·L-1VEGF),VEGF+桂皮醛(90,150μmol·L-1)组,采用管腔形成实验检测桂皮醛对VEGF诱导EA. hy 926细胞成管作用的影响;将EA. hy 926细胞分成空白组,模型组(7μg·L-1VEGF),VEGF+AG490 (50μmol·L-1)组,VEGF+桂皮醛(90μmol·L-1)组,VEGF+桂皮醛(150μmol·L-1)组,VEGF+桂皮醛(150μmol·L-1)+AG490(50μmol·L-1)组,采用蛋白免疫印迹法(Western blot)检测桂皮醛对VEGF诱导EA. hy 926细胞JAK2/STAT3通路的影响。结果:与空白组比较,模型组能够显著地促进EA. hy 926细胞增殖和迁移(P <0. 01)。与模型组比较,桂皮醛(60,90,120,150μmol·L-1)组能显著抑制VEGF诱导EA. hy 926细胞的增殖和迁移(P <0. 01)。与空白组比较,VEGF对EA. hy 926细胞成管具有一定的促进作用,成管的节点数、交叉点数、网眼数和血管分支数均有增加,但无统计学差异。与模型组比较,桂皮醛(90,150μmol·L-1)组对成管的节点数、交叉点数和网眼数均有明显抑制作用(P <0. 05,P <0. 01)。与空白组比较,模型组p-JAK2,p-STAT3,STAT3蛋白表达明显升高(P <0. 05,P <0. 01)。与模型组比较,桂皮醛(150μmol·L-1)能够显著抑制VEGF引起的p-JAK2,p-STAT3,STAT3蛋白表达升高(P <0. 01),桂皮醛(90μmol·L-1)能够显著抑制VEGF引起的p-STAT3,STAT3蛋白表达升高(P <0. 05,P <0. 01)。结论:桂皮醛对VEGF诱导EA. hy 926细胞的增殖、迁移、成管具有明显的抑制作用,该作用与抑制JAK2/STAT3通路的激活有关。
基金Supported by National Natural Science Fundation of China(No.81000387)
文摘AIMTo determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs).METHODShRVECs transfected with peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) siRNA were incubated for 24h and then placed into a normoxic (20%, O<sub>2</sub>) or hypoxic (1%, O<sub>2</sub>) environment for another 16h. PGC-1α mRNA and protein levels were detected by real-time PCR and Western blot. VEGF mRNA and protein levels were detected by real-time PCR and ELISA. Cell proliferation was evaluated by BrdU incorporation assay. Forty-eight hours after siRNA transfection, hRVECs were planted into Matrigel-coated plates and cultured under normoxic (20%, O<sub>2</sub>) or hypoxic (1%, O<sub>2</sub>) conditions for another 48h. The tube formation of hRVECs was observed under an optical microscope and quantified by counting the number of branch points and calculating the total tube length.RESULTSPGC-1α mRNA and protein levels were significantly reduced by PGC-1α siRNA, and VEGF mRNA and protein levels also decreased significantly. The percentage of BrdU-labeled cells in siPGC-1α groups were significantly decreased compared with control siRNA groups under normoxia and hypoxia in cell proliferation assay. In the tube formation assay, PGC-1α siRNA treated cells formed significantly fewer tubes.CONCLUSIONBlocking PGC-1α expression can inhibit VEGF expression in hRVECs and inhibit their ability to form tubes under both normoxic and hypoxic conditions.
文摘目的探讨映山红花总黄酮(total flavones of rhododendra,TFR)促大鼠脑血管内皮细胞体外形成血管作用及与VEGFR_(2)和神经源性硫化氢(H_(2)S)的关系。方法采用大鼠脑血管内皮细胞单独培养及和与海马神经元共培养,分别采用不同的实验方法检测细胞增殖、迁移、成管及H_(2)S含量和钙离子荧光强度,包括CCK-8法、细胞划痕法、Transwell法、基质胶成管、H_(2)S试剂盒及钙离子荧光探针法。结果在单独培养的大鼠脑血管内皮细胞上,H_(2)S供体NaHS(200μmol·L^(-1))和TFR(90、270、810 mg·L^(-1))对大鼠脑血管内皮细胞的增殖、迁移、成管及[Ca^(2+)]i荧光强度都有明显的促进作用。而VEGFR_(2)阻断剂SU5416(10μmol·L^(-1))可抑制TFR的促进内皮细胞增殖、迁移和形成血管及[Ca^(2+)]i荧光强度;在与海马神经元共培养的大鼠脑血管内皮细胞上,TFR显著地升高共培养中H_(2)S含量,并被CBS抑制剂AOAA(200μmol·L^(-1))抑制。与此同时,TFR明显地促进共培养中大鼠脑血管内皮细胞的形成血管作用,并可被AOAA和VEGFR_(2)阻断剂SU5416显著地抑制。结论TFR在体外可通过VEGFR_(2)升高[Ca^(2+)]i来促进脑血管内皮细胞形成血管,并可通过诱导神经元中CBS生成H_(2)S作用于大鼠脑血管内皮细胞的VEGFR_(2)来促进血管形成。
文摘目的:探讨微小核糖核酸(microRNA)-24(miR-24)对内皮型一氧化氮合酶(eNOS)基因表达调节的分子机制及其对血管内皮细胞增殖、迁移和管腔形成能力的影响。方法:构建miR-24及其反义序列的高表达质粒,分别转染人脐静脉内皮细胞(HUVECs),根据转染质粒将实验细胞分为:miR-24高表达组、miR-24干扰组和空白质粒对照组。用四甲基偶氮唑盐(MTT)检测HUVECs增殖能力,划痕和Transwell试验检测细胞的迁移能力,人工基底膜检测细胞的管腔形成能力;分别用逆转录-聚合酶链反应(RT-PCR)、蛋白免疫印迹法(Western blotting)检测eNOS和Sp1转录因子m RNA和蛋白表达水平。结果:(1)与空白质粒对照组比较,miR-24高表达组细胞增殖能力降低45.45%(0.36±0.04 vs 0.66±0.08,P<0.05);miR-24高表达组细胞迁移速度明显减缓,且迁移数目降低74.75%(30.25±3.78 vs 119.80±10.94,P<0.01),未能形成明显管腔样结构。(2)与空白质粒对照组比,miR-24高表达组eNOS m RNA降低46.2%(0.49±0.02vs 0.91±0.01,P<0.05),蛋白表达减少49.07%(0.55±0.05 vs 1.08±0.05,P<0.05);同时Sp1 m RNA降低44.9%(0.49±0.01 vs 0.89±0.02,P<0.05),其蛋白质表达量也相应减少54.90%(0.46±0.02 vs 1.02±0.04,P<0.05)。在miR-24抑制组中,上述指标较空白质粒对照组降低,但比miR-24高表达组显著升高,特别是小管形成数量、及管腔长度与空白质粒对照组相近。结论:miR-24显著抑制HUVECs的增殖、迁移和管腔形成的能力,并且与miR-24对eNOS的表达调控有关;miR-24明显抑制eNOS表达,Sp1的参与可能是这一调节过程的重要分子机制之一。
