绿茶多酚抑制晚期糖基化终产物诱导的大鼠血管平滑肌细胞增殖

目的 本研究观察绿茶多酚对晚期糖基化终产物(AGEs)刺激下离体大鼠胸主动脉血管平滑肌细胞增殖的影响。方法 体外培养大鼠主动脉血管平滑肌细胞(VSMCs)应用不同浓度的AGEs分别刺激VSMCs并设立对照组进行比较．将不同剂量的绿茶多酚与AGEs共同作用并设立对照组进行比较，采用非放射性的MTS／PES法确定血管平滑肌细胞的增殖状态。结果 与对照组相比，晚期糖基化终产物对血管平滑肌细胞增殖具有明显的刺激作用。在晚期糖基化终产物刺激下．绿茶多酚可明显抑制血管平滑肌细胞的生长。AGEs对p44／42MAPK磷酸化蛋白表达有显著的增强作用，此作用可被绿茶多酚抑制。结论 本研究提示绿茶多酚可抑制晚期糖基化终产物诱导的血管平滑肌细胞增殖及p44／p42 MAPK磷酸化蛋白的表达。     

绿茶多酚抑制LDL诱导的血管平滑肌细胞增殖

The proliferation and migration of vascular smooth muscle cells (VSMCs) is one of the major mechanisms of intimal thickening in atherosclerosis and post-angioplasty restenosis. Elevated plasma levels of low-density lipoprotein (LDL) have been implicated in the pathogenesis of atherosclerotic vascular diseases. The purpose of this study was to determine the effects of green tea polyphenols on the proliferation and p44/42 mitogen-activated protein kinase (MAPK) activity in rat VSMCs simulated by native LDL. Rat aortic VSMCs were cultured and treated with LDL (100 microg/ml) in the absence or presence of green tea polyphenols, and the cell proliferation was subsequently quantified by non-radioactive MTS/PES assay and the cell cycle analyzed by flow cytometry. The p44/42 MAPK activity was evaluated by immunoblotting using anti-p44/42 phospho-MAPK antibody. Compared with the cells without polyphenol treatment, the proliferation of the VSMCs induced by LDL was dose-dependently inhibited by green tea polyphenols (P<0.05), with more numerous cells in G(0)G(1) phase (P<0.05) as shown by flow cytometry analysis. LDL significantly enhanced the p44/42 MAPK activity, an effect obviously inhibited by green tea polyphenols (at 100 microg/ml). These results suggest that green tea polyphenols can inhibit high levels of LDL-induced proliferation of phosphorylated p44/42 MAPK expression in rat VSMCs. Green tea polyphenols may, therefore, offer vascular protection by inhibiting VSMC growth in response to hypercholesterolemia.     

绿茶多酚抑制LDL诱导的血管平滑肌细胞增殖

观察绿茶多酚对低密度脂蛋白(LDL)刺激下离体大鼠胸主动脉血管平滑肌细胞增殖和p44/42MAPK表达的影响。体外培养大鼠胸主动脉血管平滑肌细胞,在LDL(100 μg/ml)刺激时,应用不同剂量的绿茶多酚作用24 h后,采用MTS/PES法确定血管平滑肌细胞的增殖状态。应用流式细胞术测定细胞周期,p44/42磷酸化抗MAPK抗体的蛋白免疫印迹法测定MAPK蛋白表达。与未经LDL处理的平滑肌细胞相比,LDL可以明显刺激大鼠血管平滑肌细胞的增殖,绿茶多酚对LDL刺激下的大鼠血管平滑肌细胞增殖具有明显的抑制作用(P<0.05),并呈现剂量依赖性。流式细胞术检测表明,绿茶多酚可以使LDL刺激下的大鼠血管平滑肌细胞大部分处于G0/G1期,与LDL组相比有明显差异(P<0.05)。LDL对磷酸化p44/42MAPK蛋白表达有显著的增强作用,此作用可被绿茶多酚抑制。绿茶多酚可明显抑制LDL刺激下的大鼠血管平滑肌细胞增殖和磷酸化p44/42丝裂素活化蛋白激酶的表达。     

重组平颏海蛇磷脂酶A2抑制晚期糖基化终产物诱导的大鼠血管平滑肌细胞增殖

OBJECTIVE: To observe the effects of recombinant Lapemis hardwickii phospholipase A(2) (rPLA(2)) on vascular smooth muscle cell (VSMC) proliferation stimulated by advanced glycation end products (AGEs). METHODS: Rat aortic VSMCs were cultured in vitro and stimulated with AGEs of different concentrations prior to co-treatment with rPLA(2) and AGEs. The response of the VSMCs to these treatments was observed in comparison with that of the control group. Non-radioactive MTS/PES assay was adopted for the quantification of the cell proliferation ratio. RESULTS: Compared with the control group, AGEs significantly stimulated VSMCs proliferation (P < 0.05), but even with AGEs stimulation, rPLA(2) inhibited the growth of VSMCs (0.753+/-0.098 vs 1.100+/-0.226, P < 0.05). CONCLUSION: rPLA(2) can significantly inhibit the proliferation of VSMCs stimulated by AGEs.     

晚期糖基化终产物对大鼠血管平滑肌细胞增殖影响的实验研究

目的：研究晚期糖基化终产物（Advanced Glycation end Products,AGEs）对体外培养大鼠主动脉血管平滑肌细胞（Vascular Smooth Muscle Cells,VSMCs）增殖的影响。方法：组织贴块法培养大鼠VSMCs,给予不同浓度AGE-牛血清白蛋白（AGE-BSA）连续处理VSMCs 168 h（7 d）,每24 h采用噻唑蓝（MTT）法检测细胞增殖,绘制细胞生长曲线,计算细胞增殖促进率。结果：80 mg/L终浓度的AGE-BSA在培养72、961、20 h,10、20、40 mg/L终浓度的AGE-BSA在培养96、120、1441、68 h均可显著促进细胞增殖（P〈0.05） 40 mg/L终浓度的AGE-BSA在培养96 h时细胞增殖促进率最大（72.1%）。结论：AGEs具有促进VSMCs增殖作用,这一作用在一定范围内具有剂量依赖性。     

重组平颏海蛇磷脂酶A2抑制晚期糖基化终产物诱导的大鼠血管平滑肌细胞增殖

目的观察重组平颏海蛇磷脂酶A2(rPLA2)对晚期糖基化终产物(AGEs)刺激下离体大鼠胸主动脉血管平滑肌细胞增殖的影响。方法体外培养大鼠主动脉血管平滑肌细胞(VSMCs),应用不同浓度的AGEs分别刺激VSMCs并设立对照组进行比较,采用非放射性的MTS/PES法确定血管平滑肌细胞的增殖状态。结果与对照组相比,AGEs对VSM-Cs增殖具有明显的刺激作用(P<0.05)。在AGEs刺激下,rPLA2可明显抑制VSMCs的生长(0.753±0.098 vs. 1.100±0.226, P<0.05)。结论rPLA2可抑制晚期糖基化终产物诱导的血管平滑肌细胞增殖。     

PPARα对糖基化终产物诱导的大鼠血管平滑肌细胞的作用

目的:观察氯贝特对糖基化终产物(AGEs)刺激下的离体大鼠主动脉血管平滑肌细胞(VSMCs)增殖的作用以及对过氧化物酶体增生物激活受体α(PPARα)基因及蛋白表达水平的影响,探讨PPARα在AGEs诱导VSMCs增殖中的作用。方法:体外培养大鼠VSMCs,应用不同浓度的AGEs分别刺激VSMCs并设立对照组进行比较,采用MTT法观察不同浓度的AGEs对VSMCs增殖的影响及氯贝特与AGEs共孵育对VSMCs增殖的干预作用,并用流式细胞仪检测细胞周期,RT-PCR检测PPARα的mRNA表达,Western blotting分析PPARα的蛋白表达。结果:AGEs增加DNA的合成促进平滑肌细胞的增殖,氯贝特激活PPARα,使PPARα的表达量增加,抑制AGEs诱导的细胞增殖和DNA合成。结论:AGEs诱导平滑肌细胞增殖与平滑肌细胞PPARα表达相关,PPARα表达下调可能是产生糖尿病动脉粥样硬化的重要原因之一。     

柿叶黄酮对晚期糖基化终产物诱导的血管外膜成纤维细胞增殖的影响

OBJECTIVE: To observe the effects of flavone from the leaves of Diospyros kaki on proliferation of adventitial fibroblasts induced by advanced glycation end-products (AGEs) in vitro. METHODS: NIH/3T3 cells cultured in vitro were treated with both AGEs and flavone from the leaves of Diospyros Kaki for observation in comparison with the cells that received treatments with either AGEs or flavone, or neither. The ratio of cell proliferation was determined by non-radioactive MTS/PES assay. RESULTS: The ratio of cell proliferation was 0.840+/-0.061 in the untreated control group, and was 1.330+/-0.055, 1.210+/-0.119, 1.029+/-0.076 and 0.792+/-0.060 in AGEs groups corresponding to AGE concentrations of 100, 50, 10 and 1 microg/ml respectively. AGEs significantly induced fibroblast proliferation in a dose-dependent manner when the concentration was above 10 microg/ml (P<0.05), as compared with the untreated control group. (P<0.05). The ratio of cell proliferation was 0.829+/-0.056 in cells treated with flavone at the concentration of 50 microg/ml, which alone failed to affect fibroblast proliferation (P>0.05). With AGEs stimulation, however, flavone from the leaves of Diospyros kaki significantly inhibited the proliferation of the fibroblasts (P<0.05). CONCLUSION: Flavone from the leaves of Diospyros kaki can significantly inhibit the proliferation of adventitial fibroblasts stimulated by AGEs in vitro.     

晚期糖基化终产物对大鼠血管平滑肌细胞分泌炎症性趋化因子的影响及机制

目的 观察晚期糖基化终产物(AGE)对血管平滑肌细胞(VSMC)分泌单核细胞趋化蛋白1(MCP-1)和白细胞介素8(IL-8)的影响并初步探讨可能的细胞内信号转导机制.方法 分离、培养原代大鼠VSMC并进行鉴定.采用糖基化血清白蛋白(GSA)模拟AGE,观察不同浓度GSA(10、100和500 μg/ml)对VSMC分泌MCP-1和IL-8的影响和时间曲线;并对其进行细胞增殖率校正以消除VSMC数量增加对测定的影响;p38MAPK抑制剂(SB203580)、ERK1/2抑制剂(PD98059)及NF-κB抑制剂(PDTC)、Proteasome抑制剂(MG132)预处理后观察GSA刺激VSMC分泌MCP-1和IL-8水平的改变.结果 与空白对照组相比,100 μg/ml GSA作用24 h VSMC分泌MCP-1(13.01ng/ml±0.12ng/ml比7.02 ng/ml±0.26 ng/ml,P＜0.05)和IL-8(12.6ng/ml±0.86 ng/ml比3.07 ng/ml±0.35ng/ml,P＜0.05)水平最高.经过细胞增殖校正后,GSA仍然能够促进VSMC表达MCP-1和IL-8.MAPK抑制剂和NF-κB抑制剂预处理后发现PDTC(10 μmol/L)、SB203580(5 μmol/L)以及MG132(10 μmol/L)可以抑制GSA刺激VSMC表达MCP-1和IL-8.结论 GSA可以促进VSMC分泌致炎性趋化因子MCP-1和IL-8,这种作用独立于细胞增殖,可能是通过激活细胞内p38MAPK信号转导通路,促进核因子NF-κB的活化而实现.

Abstract：

Objective To investigate the effects of advanced glycation end products (AGEs) on the secretion of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) in vascular smooth muscle cells (VSMCs) and explore its possible intracellular signaling mechanism. Methods Primary rat VSMCs were isolated and identified. VSMCs were treated with glycation serum albumin (GSA), an important component of AGEs, in series of concentrations and time. The role of MAPK and NF-κB inhibitors was confirmed. The levels of MCP-1 and IL-8 were determined by enzyme-linked immunosorbent assay (ELISA). Results VSMCs were treated with GSA at the doses of 10 μg/ml, 100 μg/ml and 500 μg/ml respectively. In comparison with the control group, the levels of MCP-1 ( 13.01 ng/ml ± 0.12 ng/ml vs 7. 02 ng/ml ±0. 26 ng/ml, P＜0.05) and IL-8 (12. 6 ng/ml ±0. 86 ng/ml vs 3. 07 ng/ml ±0.35 ng/ml,P＜0.05) increased in the GSA-treated group, especially at the concentration of 100 μg/ml. After adjustment for cells proliferation, the levels of MCP-1 and IL-8 were still higher in the GSA-treated group.After a pretreatment of PDTC ( 10 μmol/L), SB203580 (5 μmol/L) and MG132 ( 10 μmol/L), the levels of MCP-1 and IL-8 decreased. However, it had no change when pretreated with PD98059 (20 μmol/L).Conclusion GSA promotes the secretion of MCP-1 and IL-8 in VSMCs. Such an effect is not dependent on cellular proliferation. It may be realized through an activation of NF-κB by p38MAPK-sensitive intracellular signaling pathway.     

茶多酚抑制大鼠血管平滑肌细胞增殖及相关机制研究

目的:研究晚期糖基化产物(AGEs)对大鼠血管平滑肌细胞A7R5增殖的影响及相关机制。方法:用AGEs处理A7R5细胞,相同条件BSA处理为对照组,Western blotting检测RAGE蛋白表达的变化,MTT比色法测定细胞活性。结果:AGEs处理A7R5细胞后,AGEs受体RAGE及细胞增殖水平均明显升高,但与EGCG共孵育能显著抑制AGEs诱导的RAGE表达增加及细胞增殖水平。结论:EGCG能够抑制AGEs诱导的大鼠血管平滑肌细胞增殖水平,这可能与EGCG抑制AGEs受体RAGE的表达相关。     

茶多酚对大鼠血管平滑肌细胞增殖的影响

目的　探讨茶多酚对离体大鼠胸主动脉血管平滑肌细胞增殖的影响。方法　体外培养大鼠主动脉血管平滑肌细胞 ,应用不同剂量的茶多酚作用 2 4 h后 ,采用 MTS/ PES法确定血管平滑肌细胞的增殖状态。应用流式细胞术测定细胞周期。结果　与对照组相比 ,茶多酚对血管平滑肌细胞增殖具有明显的抑制作用 (P<0 .0 0 1)。流式细胞术检测结果表明 ,茶多酚可以使血管平滑肌细胞大部分处于 G0 / G1 期 ,与对照组相比有明显差异 (P<0 .0 5 ) ,并且可以诱导其凋亡。结论　茶多酚可明显抑制离体大鼠血管平滑肌细胞的增殖。     

晚期糖化终产物对离体血管平滑肌细胞的影响

探讨晚期糖化终产物对离体新生牛胸主动脉血管平滑肌细胞的影响及机制。方法：观察ＶＳＭＣ受ＡＧＥｓ刺激后其生长,超微结构和血小板源生长因子－Ａ（ＰＤＧＦ－Ａ）的变化以及维生素Ｅ对上述变化的影响。结果：ＡＧＥｓ促进ＶＳＭＣ生长,细胞内ＡＧＥｓ和ＰＤＧＦ－Ａ的免疫反应呈阳性,线粒体和粗面内质网增多。     

二甲双胍对糖基化终产物诱导血管平滑肌细胞钙化的抑制作用

目的:在细胞水平观察二甲双胍对晚期糖基化终产物(AGEs)诱导的血管平滑肌细胞钙化的抑制作用。方法:应用大鼠血管平滑肌细胞株(A7R5)进行体外实验,分别设对照组、牛血清白蛋白(BSA)组、AGE-BSA组、二甲双胍与AGE-BSA共干预组。将200 mg.L-1 AGE-BSA与不同浓度(10-4、5×10-5、10-5 mol.L-1)的二甲双胍在含10 mmol.L-1β-甘油磷酸的钙化培养液中共培养血管平滑肌细胞,观察不同浓度二甲双胍对血管平滑肌细胞钙化的影响。采用全自动生化分析仪测定细胞层钙含量,磷酸苯二钠法检测碱性磷酸酶活性,RT-PCR检测骨形成蛋白-2(BMP-2)mRNA表达,Western blotting检测BMP-2蛋白表达。结果:与对照组相比较,AGE-BSA组细胞层钙含量增加,碱性磷酸酶活性升高,细胞BMP-2 mRNA表达升高,BMP-2蛋白表达增加(P<0.01);不同浓度二甲双胍与AGE-BSA共干预可降低AGEs诱导的上述指标升高。结论:AGE-BSA可促进血管平滑肌细胞钙化,二甲双胍可剂量依赖性地抑制AGE-BSA诱导的钙化促进作用。     

糖基化终末产物损伤冠状动脉平滑肌细胞Kv通道

目的 研究糖基化终末产物（advanced glycation end products,AGEs）对大鼠冠状动脉平滑肌细胞电压门控性钾离子（voltage-gated K⁺,K_v）通道电流的影响。方法 分离大鼠冠状动脉平滑肌细胞,分为空白对照组、糖基化牛血清白蛋白（AGE-bull serum albumin,AGE-BSA）组、AGE-BSA+抗AGE受体抗体（anti-receptor of AGEs immunoglobulin G,anti-RAGE IgG）组进行干预。采用膜片钳技术检测各组细胞K_v通道电流,采用Western blotting、实时荧光定量PCR方法检测各组细胞K_v1.2和K_v1.5通道蛋白及mRNA的表达。结果 AGE-BSA直接干预冠状动脉平滑肌细胞明显抑制了平滑肌细胞的K_v电流达32.7%,并使K_v1.2和K_v1.5通道蛋白及mRNA的表达明显下调。而给予anti-RAGE IgG预处理30 min后再加入AGE-BSA刺激,平滑肌细胞的Kv电流密度及Kv1.2和Kv1.5通道蛋白及mRNA的表达与空白对照组相比,差异无统计学意义。结论 AGEs通过结合RAGE损伤冠状动脉平滑肌细胞K_v通道。     

糖基化终产物对大鼠血管平滑肌细胞骨保护素mRNA表达的影响

目的:研究糖基化终产物(AGEs)对大鼠血管平滑肌细胞(VSMCs)骨保护素(OPG)mRNA表达的影响。方法:体外培养VSMCs,在含10 mmol.L-1β-甘油磷酸(β-GP)的培养液中加入不同浓度(0、50、100、200、400 mg.L-1)的AGE-牛血清白蛋白(BSA)与VSMCs孵育不同时间(0、12、24、36、48、72 h)。采用RT-PCR检测OPG的mRNA表达水平。结果:AGE-BSA呈时间和剂量依赖性地增加VSMCsOPG mRNA的表达;200 mg.L-1的AGE-BSA与VSMCs孵育24 h,OPG mRNA表达水平达到高峰。结论:AGE-BSA能够促进大鼠VSMCsOPG mRNA的表达。     

重组海葵溶细胞素对大鼠血管平滑肌细胞增殖的影响

目的观察重组海葵溶细胞素(Src)对离体大鼠血管平滑肌细胞增殖的影响。方法体外培养大鼠胸主动脉血管平滑肌细胞(VCMC),应用不同浓度的Src分别进行刺激VCMC并设立对照进行比较,采用非放射性的MTS/PES法确定血管平滑肌细胞的增殖状态。结果各组的细胞增殖率分别为对照组:0.802±0.055;Src100μg/ml组:0.115±0.014;Src10μg/ml组:0.213±0.016;Src1μg/ml组:0.335±0.097,Src100ng/ml组:0.663±0.060;Src10ng/ml组:0.678±0.129;Src1ng/ml组:0.738±0.072。统计分析显示100ng/ml以上浓度的Src能明显抑制大鼠血管平滑肌细胞的增殖(P<0.05),并呈现剂量依赖性(P<0.05)。结论重组海葵溶细胞素Src对大鼠血管平滑肌细胞增殖有一定的抑制作用。