【作者】 张勇；

【导师】 孙连坤；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 有研究证明,受体介导的配体内吞可以降低细胞膜表面受体的数量,使受体配体复合物进入胞内溶酶体降解并发生解离,引起受体功能的下调和再循环至细胞膜表面。另外,还有学者认为内吞进入细胞内的配体受体复合物可能参与了下游信号的转导与调控,发挥其生理学及病理生理学作用。血管紧张素II(Angiotensin II,Ang II)是肾素-血管紧张素系统(Renin-angiotensin system,RAS)最主要的效应分子,具有收缩血管效应、调控醛固酮分泌等生理功能。Ang II与血管紧张素II 1型受体(Angiotensin II 1 type receptor,ATl receptor)在细胞膜表面结合,一方面激活下游信号转导途径发挥生物学作用,另一方面快速的以网格蛋白包被小泡的方式发生内吞,其中组成网格蛋白包被的主要衔接蛋白为衔接蛋白复合体2(AP2)。最近的研究表明,AngII与细胞膜表面AT1受体结合,刺激心肌细胞肥大和(或)成纤维细胞增生,是导致心肌肥厚的主要原因之一。但是,ATl受体介导的AngⅡ内吞是否也参与了心肌肥厚的形成尚需进一步地研究。本实验利用流式细胞术、激光共聚焦显微镜、RNAi、RT-PCR及Western-blot等技术,研究发现AngII可以引起H9C2细胞体积增加;p38的磷酸化形式增多,NF-κB(p65)入核增多和氯离子通道蛋白ClC-2表达增加;同时使细胞进入S期的比例增加,G1期的比例降低,提示细胞DNA和蛋白合成增加。应用内吞特异性抑制剂PAO可以抑制上述细胞体积和信号途径的改变,与应用AT1受体拮抗剂(ARB)candesartan的作用一致,即PAO与Candesartan都可抑制AngII的促细胞肥大增生作用。初步证明AngII在细胞膜上与AT1受体结合后,在激活下游心肌肥大相关信号因子的同时,发生快速的受体内吞,这种配体介导的受体内吞在进入细胞内可能依然发挥着病理生理学作用。本实验又通过RNAi技术敲除AP2,抑制网格蛋白介导的内吞,发现可以抑制AT1受体介导的AngII内吞,并影响细胞体积,细胞周期及相关信号蛋白的改变,但对ClC-2表达无明显影响。因此我们得出初步结论,ATl受体介导的AngII内吞,不但使配体受体复合物进入细胞内,发生降解和受体配体的解离,及受体的再循环,而且内吞入胞内的受体在细胞内继续发挥其生物学功能,持续激活下游信号,通过影响细胞周期的改变,引起细胞的肥大增生。但其具体作用机制还需要进一步研究探讨。更多还原

【Abstract】 ObjectiveMyocyte hypertrophy can increase the risk of cardiac ischemia, left ventricular dysfunction and congestive heart failure, and it is a risk factor of leading to sudden cardiac death. Angiotensin II(Ang II) is the most important effect molecule of myocyte hypertrophy in renin-angiotensin system. Ang II binding its angiotensin II type 1(AT1) receptor in cell surface, can active downstream signal transduction pathways, and stimulate myocyte hypertrophy and/or fibroblast proliferation , leading to cardiac hypertrophy. While Ang II active AT1 receptor, the ligand and receptor complax endocytosis by clathrin-coated vesicle. However , the role of model, pathway and signal transductiong of AT1 receptor-mediated endocytosis in cardiac hypertrophy formation is not clear. Previous studies suggest that endocytosis can decrease the number of receptor of cell surface, promote degradation of receptor ligand complex in lysosome, and cause receptor function down-regulation and receptor recycling. However, some people have found, the ligand receptor complex through endocytosis can continue to active downstream signals, play a role of pathophysiology. Therefore , based on the view, we inhibited the AngII AT1 receptor-mediated endocytosis by endocytosis inhibitors ((phenylarsine oxide, PAO) and RNAi endocytosis-related protein (adaptor protein 2, AP2), observed the AngII induced myocardial cell hypertrophy and hyperplasia associated signaling pathway for the pathogenesis of cardiovascular diseases and provided a theoretical basis for clinical treatment.Methods:By rat myocardial cell line (H9C2), through AngII, endocytosis inhibitor (PAO), angiotensin II receptor blocker (candesartan) and RNAi silencing endocytosis-related protein(AP2), observed the following points:(1) H9C2 cells treated with candesartan and the PAO 30min, then added Ang II 24h, cells were collected and analyzed cell volume changes by flow cytometry.(2) H9C2 cells treated with candesartan and the PAO 30min, then added AngⅡ30min, cells were collected and extracted total protein, cytoplasmic and nuclear proteins from the cells, observed the changes of phosph-p38 and the distribution of NF-κB (p65) in cytoplasm and nucleus.(3) H9C2 cell synchronized by serum starvation, treated with candesartan and the PAO 30min, then added Ang II 24h, PI staining, detected change of cell cycle (G1, G2, S and M phase) by flow cytometry.(4) Construction of AP2 interference vector and detected silence effect. According to rat AP2-μ2 subunit gene sequences from Gene Bank and RNAi design principles, we designed and constructed three pSH1Si-AP2 expression vector. By transfecting H9C2 cells, silence effect of AP2 mRNA and protein expression was detected by RT-PCR and Western Blotting.(5) H9C2 cells treated with AngII at different time points (5min, 10min, 15min and 30min) and at different concentrations (10-6M ,10-7M and 10-8M), the morphology change of AT1 receptor-mediated endocytosis of AngⅡwere detected by the immunofluorescence technique and laser confocal microscope.(6) H9C2 cells treated with PAO, candesartan, and transfected pSH1Si-AP2 plasmid, the morphology change of AT1 receptor-mediated endocytosis of AngⅡwere detected by laser confocal microscope.(7) The AngII-induced cell volume, p38 phosphorylation, NF-κB (p65) of nuclear transfer and cell cycle changes was observed by inhibiting the expression of AP2 protein of H9C2 cells. Results:Testing cell survival by MTT, H9C2 cells survival had no effect at PAO 0.25μM and candesartan 40μM, so we choose the drug dose of above in experiment. H9C2 cell treated with Ang II. AngII also induced activation of MAPK signaling pathway throught p38 phosphorylation; and allowed NF-κB (p65) moving into the nucleus. Candesartan can inhibit the above change and decrease p38 phosphorylation, p65 nuclear transfer reduction by inhibiting Ang II and AT1 receptor binding at cell membrane. PAO also inhibited Ang II-induced p38 phosphorylation and p65 nuclear translocation. Cell cycle changes were observed in H9C2 cells treated with Ang II that G0/G1 phase cells decreased, and G1 / S phase cells increased by flow cytometry. Candesartan and PAO can inhibit above change of cell cycle.AP2 RNAi expression vector was successfully constructed by sequencing non-base mismatch and loss. pSH1Si-AP2 plasmid-1 plasmid can inhibit the AP2 of mRNA and protein expression by transfected AP2 RNAi expression vector in H9C2 cells. It suggested that AP2 RNAi vectors can be successfully silence the expression of AP2. After treating with AngII, AT1 receptor endocytosis quickly occur by immunofluorescence in H9C2 cells, and the most efficient endocytosis occur in Ang II concentration of 10-7M for 15min. Candesartan and PAO can inhibit AT1 receptor endocytosis. PSH1Si-AP2 plasmid-1 can also inhibit the AT1 receptor-mediated Ang II endocytosis after transient transfection of H9C2 cells, suggesting that endocytosis of Ang II and AT1 receptor mainly by clathrin-coated vesicles. At the same time, AP2 silence can suppress cell volume increase induced by Ang II, p38 phosphorylation and p65 nuclear transfer, also inhibit Ang II-induced cell cycle changes.Conclusion:(1). By flow cytometry, Ang II can increase the volume of H9C2 cells, successed copy the model of cell hypertrophy and hyperplasia. Candesartan and PAO can inhibit the AngII role in promoting cell hypertrophy. Candesartan for ligand binding receptor antagonist, directly block the downstream signal transduction, inhibition of cell hypertrophy, PAO mainly endocytosis inhibitors, considered by blocking endocytosis , affected downstream signal transduction, and inhibition of cell hypertrophy.(2). Ang II induced H9C2 cell activation of MAPK signaling pathway, increased p38 phosphorylation and NF-κB (p65) into the nucleus. Candesartan can inhibit the activation of these signaling pathways, by inhibiting the AT1 receptor and Ang II binding at membrane, it suggested that Ang II promotes H9C2 cells hypertrophy by activating p38 sigal and affecting the NF-κB (p65) into the nucleus.(3). PAO also can inhibit Ang II-induced p38 phosphorylation and p65 nuclear transfer, because PAO was the inhibitor for the AT1 receptor endocytosis, it suggested that inhibiting the AT1 receptor-mediated Ang II endocytosis may also prevent the MAPK and activation of NF-κB pathway, endocytosis of the ligand receptor complex is likely to continue the signal transduction pathway.(4). Ang II can decrease the proportion of H9C2 cells of G0/G1 phase cells, and increased the proportion of G1 / S phase, suggesting that Ang II promote DNA and protein synthesis by influencing the cell cycle, causing cell hypertrophy and hyperplasia. Similar with candesartan, PAO also can inhibit change of the cell cycle, suggesting that inhibition of receptor endocytosis also affected the changes of cell cycle by Ang II induced.(5). After treating with AngII, AT1 receptor endocytosis quickly occur by immunofluorescence in H9C2 cells, and the most efficient endocytosis occur in Ang II concentration of 10-7M for 15min. Candesartan can inhibit AT1 receptor endocytosis by inhibiting ligand and receptor binding. PAO also inhibit endocytosis by inhibitiong of receptor intracellular domain tyrosine kinase phosphorylation.(6). pSH1Si-AP2 plasmid-1 can also inhibit the AT1 receptor-mediated Ang II endocytosis after transient transfection of H9C2 cells, suggesting that endocytosis of Ang II and AT1 receptor mainly by clathrin-coated vesicles. At the same time, AP2 silence can suppress cell volume increase induced by Ang II, p38 phosphorylation and p65 nuclear transfer, also inhibit Ang II-induced cell cycle changes, suggesting that the ligand receptor complex endocytosis into the cells may also be involved in activation of downstream signal transduction pathways, and continue to play its biological role.Ang II and AT1 receptors, through activation of MAPK and NF-κB signal transduction pathways, affect the cell cycle changes, and promote cell hypertrophy and hyperplasia; PAO and AP2 RNAi inhibited AT1 receptor-mediated Ang II endocytosis ,at the same time, affected the MAPK and NF-κB pathway activation and cell cycle changes, inhibited the AngII role in promoting cell hypertrophy and hyperplasia, indicating that AT1 receptor-mediated Ang II endocytosis process may also continue to activate downstream signal transduction pathways, involved in the formation of myocardial hypertrophy mechanism.更多还原