论文标题:MAPKs及PPARs在心肌肥大和心力衰竭发生和防治中的作用和细胞机理
Effects and Cellular Mechanisms of p44/p42MAPKs and PPAR-α/-γ on the Progression and Prevention-treatment of Myocardial Hypertrophy and Heart Failure
论文作者 张世勤
论文导师 周宏灏；李云霞,论文学位 博士,论文专业 药理学
论文单位 中南大学,点击次数 546,论文页数 99页File Size4444k
2003-05-01论文网 http://www.lw23.com/lunwen_216097/ 心肌肥厚;心衰;丝裂素活化蛋白激酶;过氧化物酶体增殖物激活受体;血管紧张素II;去甲肾上腺素;肿瘤坏死因子-α;反义寡核苷酸;心肌细胞;细胞培养;大鼠
myocardial hypertrophy,heart failure,mitogen-activated protein kinase,peroxisome proliferator-activated receptor,angiotensin II,norepinephrine,tumor necrosis factor-α,antisense oligodeoxynucleotide,cardiac myocyte,cell culture,rat
MAPKs及PPARs在心肌肥大和心力衰竭发生和防治中的作用和细胞机理 已经知道心肌细胞(cardiac myocyte，MC)肥大是由多种神经内分泌因子(如AngⅡ，NE和ET-1)引起的细胞生长异常(过度生长)；而心肌过度表达炎性细胞因子TNF-α通过引起MC凋亡是导致心肌演变为衰竭的关键步骤。因此，研究调控和影响MC生长和TNF-α生成分泌的细胞机制有助于阐明心肌肥厚和心力衰竭的发病机理和探寻新的防治措施。 近年研究显示：MAPKs和PPARs是细胞生物学活动和细胞分泌功能的重要调节机构。MAPKs是存在于细胞胞浆的蛋白激酶超家族(主要亚家族为p44/p42MAPK和p38MAPK)，是细胞外信号诱导的生长增殖反应和凋亡反应的信号转导通路中具有汇聚功能和越核膜转导作用的关键信号分子。PPARs(含PPAR-α和γ两个亚型)是由配体激活的转录因子，属Ⅱ型细胞核受体。近年在免疫细胞或平滑肌细胞上发现，PPARs具有抗炎活性和抑制细胞增殖的功能，而且在MC有PPAR-α和-γ亚型的丰富表达。但这两者与心肌肥大和心衰的关系尚很少了解。 据此，本论文(1)在培养MC标本上，以其蛋白质合成速率(~3H-亮氨酸掺入法)、蛋白总量、细胞直径和/或生长相关性原癌基因c-fos和c-myc mRNA丰度(Northern blot法)作为反映MC生长水平和/或p44/42MAPK下游核底物表达的指标。(2)研究p44/p42MAPK和/或PPAR-α/-γ对AngⅡ和NE两种活性因子诱导的MC肥大和心力衰竭发生和防治中的作用和机理。 本论文包括4个分题，主要实验结果如下：(1)AngⅡ(10nmol/L)或NE(1μmol/L)可引起MC肥大反应和p44/p42MAPK活性水平增高(Western blot法测定p44/p42MAPK磷酸化蛋白含量反映活性水平)；AngⅡ或NE激活p44/p42MAPK的受体(R)-PKC信号通路各不相同，前者由AngⅡⅠ型R(AT_1R)-PKC通路介导；后者由β-肾上腺素R-PKA和α1-肾上腺素R-PKC两条通路介导；而阻断上游激酶对p44/p42MAPK的激活，两种因子诱导的MC肥大反应均明显受到抑制。(2)进一步采用反义ODN技术的研究表明，脂质体法向MC转染p44/p42MAPK反义ODN，AngⅡ诱导的p44/p42MAPK激活、c-fos和c-myc mRNA的上调以及MC肥大反应均受到明显抑制，而不受正义ODN和随机ODN的影响。(3)PPAR-α博士学位论文 中文摘要亚型激活剂（实验性调脂化合物）或 PPAR个亚型的天然或药物激活剂O 干GJ。或胰岛素增敏齐ciglitazone）处理MC，丁。上训j巨Angll或NE诱导下MC肥大反I仕。（4）TNF．a可致MC调亡：MC无TNF－。的基础分泌（ELISA法测MC培养液小TNF《含量，粘度为spg／ml人细菌脂多糖（可引起急性。0衰）促使TNF呕生成达77．spg／pl雪 而PPAN一a或1激亏剂处工均明 显引周树脂8柳一TNF一a高表达；此种负调控作用后被＊PA卜OU1失亏 剂所阻断；＊N卜o、 引起＊C凋1‘：且p38M＊PK hi性水平增高，Kp38MA卜K八＊N卜＊诱导的MC凋上：小起保力仆J！工 综上可见：＊）①p44／p42MMK是Angl！或NE两种“致肥大”因子诱导的＊C肥大反应的共同信号转导通路：柳叩巾ZM＊rK的激活是肥大反应发生的关键信号机制，阻断剂或反义ODN下调p44…42MAPK的活性，可抑制MC肥大反应。口P PARPAR记和1的激活对两种因子诱导的肥大反应和MC＊＊卜。生成只负调控作用。O）反义0＊N和P PAPA＊一体Y激活剂（调脂药和胰岛素增敏剂）只有防治心肌肥厚和。0衰的应用前景。们pM他的作川和机现何利进·步研究。。
It is well known that cardiac myocyte(MC) hypertrophy induced by a variety of bioactive agents(mainly Angll, NE, ET-1) is considered as an abnormal excessive growth of MC. Meanwhile, Overexpression of inflammatory cytokine TNF-α(a potent inducer of MC apoptosis) in MC is a key step for progression of heart failure(HF). Therefore, to investigate the cellular mechanisms responsible for controlling and regulating the growth and TNF-a production in MC might be helpful to clarify the pathogenesis and to develop new intervention methods for prevention-treatment of myocardial hypertrophy(MH) and HERecently, a variety of studies have indicated that MAPKs and PPARs(two isoforms -a and -y) play an important role in regulation of cell biological process and secretion function. MAPKs are the cytoplasmic signaling molecular family that can converge and transduct extracellular signals into cell nucleus for mediating the cell biological responses such as growth and proliferation(by the subfamily p44/p42 MAPK) and apoptosis(by p38MAPK). PPAR-KX/-Y, belonging to type II nuclear receptor subfamily, are ligand-dependent transcription factors. It has recently been demonstrated that PPAR-@/-γ play an important role in regulating the inflammatory response in macrophages and proliferation in vascular smooth muscle cells. But the effects of p44/p42MAPK and PPAR-α/-γ on MH and HF are still unclear. Thus, in this study, we designed (1)﹖o use MC protein synthesis rate(3H-leucine incorporation assay), total protein content, cell diameter and/or c-fos mRNA and c-myc mRNA abundance(by Northern blot method) as the indexes of cell growth state. (2)to study the effects of activation and deactivation of p44/p42MAPK and PPAR-α/-γ on Angll and NE-induced MC hypertrophic responses and/or on LPS-induced MC TNF-α production. (3)to clarify the pathogenesis in progression of MH and HF, and to provide new targets and develop effective agents to prevent and treat MH and HF.This study consists of 4 parts, results in each part as follows: (1)Treatment ofMC with AngII(10nmol/L) and NE(1μmol/L) resulted in elevation of growth index (considered as hypertrophic response, HR), activation of p44/p42MAPK (assayed by phospho-p44/p42MAPK protein content, Western blot method). When the activation of p44/p42MAPK was blocked by using U0126(a MEK-MAPK pathway blocker), both the Angll-induced and NE-induced HR were inhibited. On the other hand, the receptor-signal transduction pathway for Angll or ME to activate p44/p42MAPK is quite different, suggesting that p44/p42MAPK is the common signaling pathway for HR induced by both Angll and NE. (2)Angll-induced activation of p44/p42MAPK, HR and upregulation of c-fos mRNA and c-myc mRNA were all inhibited by transfection of MC with antisense oligodeoxynucleotide(ODN) to p44/p42MAPK. (3)There was no basal TNF-α production from MC. Incubation MC with LPS(a potent agent causing acute HF), TNF-α production (assayed by ELISA method) increased significantly. (4)both the Angll- and NE-induced HR as well as LPS-induced TNF-α production were inhibited after activation of PPAR-a or -y by corresponding ligands.In conclusion, the present study demonstrates that (1)activation of p44/p42MAPK is the most important signaling mechanism for progression of MC hypertrophic response, (2)activation of PPAR-o/-y functions as a negative regulator for the MC hypertrophic response as well as TNF-a production, (3)antisense ODN to p44/p42MAPK and PPAR-o/-y activators(such as lipid modulator and insulin sensitizer) may exert direct anti-hypertrophy and anti-HF effects, (4)effect of PPARs on MH and HF and their underlying mechanisms need further investigation.
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