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real-time RT –PCR 结果以较Control 组增加倍数(mean±SEM).表示，以GAPDH 为内参。(*P<0.05 与control 组比较, †P<0.05 与Ang II 组比较).B: 各组VSMCs PPAR-γ 蛋白表达情况.Westernblot 结果以较Control 组 增加倍数(mean±SEM).表示，以β-actin 为内参，。(*P<0.05 与control 150 组比较, †P<0.05 与Ang II 组比较)。 Figure 1 Effect of Ang II and rosiglitazone on VSMCs PPAR-γ expression. A: Real-time RT-PCR results are expressed as fold increase over control (mean±SEM). Data from 3 independent experiments are shown. GAPDH was served as an internal control (*P<0.05 vs. Control, †P<0.05 vs. Ang II).B: A representative immunoblots for PPAR-γ and β-actin from 3 separate experiments are shown. PPAR-γ protein expression is shown as fold increase 155 over control. Results are presented as mean±SEM. β-actin was used as an internal control. 图2 Ang II 及rosiglitazone 对VSMCs PPAR-γ 活性的影响。以DNA-binding 分析检测PPAR-γ 活性，NSB 表示非特异性结合，C1 表示竞争性结合，PC 表示阳性对照。结果以OD450 吸光度表示，(*P<0.05 与control 160 组比较, †P<0.05 与Ang II 组比较)。 Figure 2. PPAR-γ activation was analyzed by DNA-binding assay using PPAR-γ transcription assay kit, where NSB indicates non-specific binding, C1 for competitor, and PC for positive control. Results are mean±SEM of 3 independent experiments, expressed as OD 450 (*P<0.05 vs. Control, †P<0.05 vs. Ang II). 图3 PPAR-γ 激活对VSMCs Col III、FN 以及CTGF 表达的影响。以不同浓度rosiglitazone（1.0×10－6、5.0×10 －6、10.0×10－6 mol/L）预处理VSMCs 1 小时后，加入Ang II (1.0×10－7 mol/L)刺激24 小时，用real-time RT-PCR 方法检测VSMCs 中ColIII、FN 及CTGF 表达情况。结果以较Control 组增加倍数(mean±SEM).表示，以 GAPDH 为内参。(*P<0.05 与control 组比较, †P<0.05 与Ang II 组比较). 170 Figure3. Rosiglitazone inhibited Col III, FN and CTGF mRNA expression in Ang II-stimulated VSMCs. Cells were pretreated with increasing concentrations of rosiglitazone （1.0×10－6、5.0×10－6、10.0×10－6 mol/L）for 1 hour and subsequently stimulated with Ang II (1.0×10－7 mol/L) for 24 h. (A), real-time RT-PCR revealed that rosiglitazone inhibited Ang II-induced CTGF, FN and CTGF mRNA expression in VSMCs. Results are expressed as fold increase over control and mean±SEM data of 3 independent experiments are shown. GAPDH was served as 175 an internal control (*P<0.05, vs. Control, †P<0.05, vs. Ang II). 图4 PPAR-γ 激活对VSMCs 蛋白表达及分泌的影响。以不同浓度rosiglitazone（1.0×10－6、5.0×10－6、10.0×10 －6 mol/L）预处理VSMCs 1 小时后，加入Ang II (1.0×10－7 mol/L)刺激24 小时。以westernblot 检测VSMCs 中及细胞培养液中CTGF 含量。结果以较Control 组增加倍数(mean±SEM).表示，以β-actin 为内参。(*P<0.05 与control 组比较, †P<0.05 与Ang II 组比较)。 185 Figure 4. Rosiglitazone inhibited CTGF protein expression and secretion in Ang II-stimulated VSMCs. concentrations of rosiglitazone （1.0×10－6、5.0×10－6、10.0×10－6 mol/L）for 1 hour and subsequently stimulated with Ang II (1.0×10－7 mol/L) for 24 h. Western blots documented that rosiglitazone significantly inhibited Ang II-induced CTGF protein secretion and production in VSMCs. All results are representative of 3 independent experiments. β-actin in cell fragment was served as an internal control. All values are showed as mean±SEM of 3 190 experiments (*P<0.05, vs. Control, †P<0.05, vs. Ang II). 图 5. PPAR-γ 活化参与了rosiglitazone 对Ang II 诱导的CTGF 蛋白过表达的抑制作用. 用或不用PPAR-γ 拮抗剂GW9662 (3 ×10－6 mol/L) or BADGE (195 1×10－6 mol/L)预先处理细胞30 min 后分别加 入rosiglitazone (5 ×10－6 mol/L), 15-d-PGJ2 (5 ×10－6 mol/L) or pioglitazone (50 ×10－6 mol/L)1 小时，最后加入 Ang II (1 ×10－7 mol/L)刺激24 小时。分别用real-time RT-PCR 法及westernblot 法检测CTGFmRNA 及蛋白 表达情况。结果以较Control 组增加倍数(mean±SEM).表示， (*P<0.05 与control 组比较, †P<0.05 与Ang II 组比较，#P<0.05 与 Ang II + Ros 组比较)。 200 Figure 5 Suppression of CTGF expression is mediated by PPAR-γ. Cells were pretreated with or without GW9662 (3 ×10－6 mol/L) or BADGE (1×10－6 mol/L) for 30 min prior to the addition of rosiglitazone (5 ×10－6 mol/L), 15-d-PGJ2 (5 ×10－6 mol/L) or pioglitazone (50 ×10－6 mol/L) for 1, and subsequently stimulated with Ang II (1 ×10－7 mol/L) for 24 h. A Real-time RT-PCR Results are expressed as fold increase over control and mean±SEM data of 3 independent experiments are shown. GAPDH served as an internal control (*P<0.05 vs. Control, 205 †P<0.05 vs. Ang II, #P<0.05 vs. Ang II + Ros). B. A representative Western blotting (upper panel) and mean±SEM data (bottom panel) of 3 independent experiments are shown. Results are expressed as fold increase over control. β-actin served as an internal control (*P<0.05 vs. Control, †P<0.05 vs. Ang II, #P<0.05 vs. Ang II + Ros). 210 表 1 real-time RT-PCR 扩增引物 目标基因 上游引物 下游引物 扩增片段长度 PPAR-γ 5’-TGGAG-CCTAA-GTTTG-AGTTT G-3’ 5’-ATCTT-CTGGA-GCACC-TTGG-3’ 226 bp CTGF 5’-AAGAA-GACTC-AGCCA-GACC-3’ 5’-AGAGG-AGGAG-CACCA-AGG-3’ 260 bp Col III 5’-AGATG-CTGGT-GCTGA-GAAG-3’ 5’-TGGAA-AGAAG-TCTGA-GGAAG-G-3’ 134 bp FN 5’-GTGAA-GAACG-AGGAG-GATGT-G-3’ 5’-GTGAT-GGCGG-ATGAT-GTAGC-3’ 267 bp 表2 各组VSMCs 上清培养液中羟脯氨酸含量 Table 2 Hydroxyproline concentrations in the culture medium of each group groups 羟脯氨酸含量 Control 5.95±0.72 Rosiglitazone(5.0×10-6) 5.84±0.69 Ang II 9.6±0.97* Ang II+Rosiglitazone (1.0×10-6) 7.9±0.89† Ang II+Rosiglitazone (5.0×10-6) 7.1.±0.88† Ang II+Rosiglitazone (10.0×10-6) 6.3.±0.81† Ang II+ Ang II+Rosiglitazone+GW9662 8.8±0.91# *P<0.05, vs. Control, †P<0.05, vs. Ang II, #P<0.05, vs.Ang II+Ros 215 3 讨论 高血压及糖尿病是导致冠状动脉疾病及粥样斑块形成的主要危险因素[5]。而血管纤维 化是高血压及糖尿病患者血管的主要组织学改变之一[9, 10]。在本研究中，我们发现PPAR-γ 激活可改善Ang II 诱导的CTGF 表达及ECM 释放。提示PPAR-γ 在血管纤维化进程中起重 220 要作用的认识，并为以PPAR-γ 为靶点防治血管纤维化提供了新的分子生物学证据。 研究表明，在VSMCs 中，Ang II 可诱导ECM 合成[11]。本部分研究通过在体实验及体 外研究均进一步证实了这一点，更为重要的是我们首次通过体外实验和在体研究证实 PPAR-γ 激动剂rosiglitazone 可在VSMCs 中抑制Ang II 诱导的胶原及非胶原ECM 释放。我 们的实验结果与Fu 等人的研究发现有相似之处。他们的研究表明离体情况下，PPAR-γ 激 225 活可抑制TGF-β1 诱导的FN 表达[12]。 结缔组织生长因子是一种潜在的致纤维化因子，它在皮肤疾病、肿瘤进展、肺纤维化以 及肾脏疾病中起重要作用。在心血管系统，CTGF 在斑块组织内、心肌梗死组织及缺血的心 肌组织[13]，以及高血压动物血管组织内高表达[14]。提示CTGF 可能是血管纤维化发生的 关键分子。我们的研究表明，在体外培养的VSMCs 中，Ang II 可促进CTGF 的表达及旁分 230 泌，同时抑制PPAR-γ 与PPRE 的DNA 结合活性。对VSMCs 进行rosiglitazone 预处理可显 著上调PPAR-γ 的表达及DNA 结合活性，同时抑制CTGF 的产生及旁分泌。此外通过应用 PPAR-γ 拮抗剂GW9662 及BADGE，本研究进一步证实了PPAR-γ 的激活参与了rosiglitazone 对Ang II 诱导CTGF 表达的调节作用。这些结果清楚地表明PPAR-γ 激活可抑制Ang II 诱 导的CTGF 表达及ECM 沉积，提示PPAR-γ 在Ang II 诱导的血管纤维化中可能起重要作用， 235 因为Ang II 在诱导ECM 沉积及CTGF 表达的同时可显著抑制PPAR-γ 活性。而当应用外源 性激动剂激活PPAR-γ 后，ECM 沉积及CTGF 表达均显著降低。有趣的是，我们同时发现 PPAR-γ 拮抗剂GW9662 及BADGE 并不能完全拮抗rosiglitazone 对Ang II 诱导CTGF 表达 的抑制作用。提示其它非PPAR-γ 途径亦可能参与了rosiglitazone 对Ang II 诱导CTGF 表达 的抑制作用。既往研究表明，人工合成的另一种PPAR-γ 激动剂pioglitazone 可通过激活 240 NF-κB 及 AP-1 途径，调节ECM 的释放[15]。因此我们推测其它诸如NF-κB 及 AP-1 等信 号转导通路亦可能参与了rosiglitazone 对Ang II 诱导的CTGF 表达及ECM释放的调节作用。 4 结论 综上所述，本部分研究证实，在VSMCs 中， PPAR-γ激活可抑制Ang II 诱导的CTGF 表达，同时抑制ECM 沉积。该结果提示我们，PPAR-γ可能在血管纤维化中起重要作用， 245 PPAR-γ激动剂或可用于预防血管纤维化的发生。 学术论文网Tag：代写论文 论文发表 代发论文 职称论文发表 医学论文 |
