【作者】 董雪；

【导师】 郭秀丽；

【作者基本信息】 山东大学 ， 药理学， 2011， 硕士

【摘要】 血管平滑肌细胞(vascular smoooth muscle cell, VSMC)位于动脉血管中膜,是血管中层唯一的细胞成分。VSMC的异常增殖,导致其由中膜迁移到内膜下间隙,是冠状动脉硬化、经皮穿刺腔内冠状动脉成形术再狭窄以及高血压血管肥厚等疾病的病理基础。因此找到VSMC异常增殖的机制,抑制VSMC异常增殖是治疗这些血管肥厚性疾病的重要途径。人第10号染色体缺失的磷酸酶及张力蛋白同源的基因(phosphatase and tensin homolog deleted on chromosome ten, PTEN)是近年来发现的一种新的抑癌基因,是具有双重磷酸酶活性的肿瘤抑制因子,在中枢神经系统、心脏、肝脏、肾脏、胃肠道、肺脏及皮肤等全身多器官均有表达,且参与多种生理过程,尤其是对肿瘤细胞的生长、增殖、分化、凋亡、粘附和迁移均具有重要影响。近年来,对PTEN的研究逐渐从肿瘤领域延伸到其他领域中,随着研究的深入人们发现PTEN在心肌肥大,高血压,动脉粥样硬化,支气管哮喘哮等疾病中也发挥重要的作用。本课题应用血管紧张素Ⅱ(AngiotensinⅡ, AngⅡ)诱导血管平滑肌细胞增殖和迁移,从细胞和分子水平深入探讨AngⅡ是否影响平滑肌细胞中抑癌基因PTEN的活性和表达以及其下游信号通路,发挥促血管平滑肌细胞异常增生和迁移的作用。并且初步探讨血管平滑肌细胞增殖中PTEN的表达如何被调控。一、血管紧张素Ⅱ促进平滑肌细胞增殖和迁移,抑制凋亡应用MTT法,CCK-8法及划痕实验,证实AngⅡ可以促进平滑肌细胞的增殖与迁移,并且确定了AngⅡ的最佳浓度和作用时间。经MTT法检测细胞活力,发现1μmol/L以及10μmol/L两个浓度的AngⅡ作用12小时和24小时后均能显著促进平滑肌细胞增殖,而0.1μmol/L AngⅡ作用不及另两个浓度。1μmol/L AngⅡ增殖促进率为12小时13.6%(p<0.001),24小时13.9(p<0.01),作用稳定。再经CCK-8法确证1μmol/L的AngⅡ在24小时内呈时间依赖性的促进平滑肌细胞增殖,12小时和24小时对平滑肌细胞增殖的促进率分别为10.4%(p<0.01)和9.8%(p<0.05)。划痕实验的结果显示,1μmol/L的AngⅡ在12小时内呈时间依赖性的促进平滑肌细胞迁移,12小时后AngⅡ组平滑肌细胞迁移的距离约为正常组的1.7倍(p<0.05)。在检测AngⅡ影响细胞活力的同时,应用免疫印记法检测了细胞中caspase-3的表达,来反映细胞凋亡水平。结果显示,12小时内1μmol/L的AngⅡ可使平滑肌细胞中Caspase-3蛋白的表达呈时间依赖性的减少,降低细胞的凋亡水平。二、血管紧张素Ⅱ对平滑肌细胞中PTEN及其通路的影响实验应用western blot检测法及非还原电泳法检测AngⅡ刺激细胞后,PTEN, phospho-PTEN (p-PTEN), reduced-PTEN (re-PTEN), oxidized-PTEN (ox-PTEN)及PTEN下游通路蛋白phospho-Akt (p-Akt)和phospho-FAK (p-FAK)表达的变化。同时应用Realtime-PCR法检测PTEN mRNA水平的变化。此外,应用还原型二氯荧光素(2’,7’-Dichlorofluorescin diacetate, DCFH-DA)检测细胞内活性氧(reactive oxygen species, ROS)水平的变化,考察自由基水平与re-PTEN和ox-PTEN表达的关系。结果显示,0.1μmol/L, 1μmol/L以及10μmol/L的AngⅡ分别作用于血管平滑肌细胞24h时,细胞内PTEN蛋白的表达水平呈浓度依赖性降低。选用1μmol/L的AngⅡ作用于平滑肌细胞0-12h, PTEN的表达呈现时间依赖性的降低,12h时,其表达降低至正常对照组的1/4(p<0.001)o RT-PCR的检测结果显示lμmol/L　AngⅡ刺激细胞2h后PTEN mRNA水平已降低至对照组的1/2(p<0.01)。说明AngⅡ可以从基因和蛋白水平降低PTEN的转录和表达。应用western blot方法检测到,AngⅡ刺激平滑肌细胞5min后可快速却短暂的升高p-PTEN的表达,2小时后趋于正常水平。向细胞内载入DCFH荧光探针,用Ang II刺激后不同时间检测到的细胞内荧光强度反映细胞内活性氧水平。结果显示,AngⅡ刺激平滑肌细胞可以快速升高细胞内活性氧的水平,1小时后达到对照组的1.5倍(p<0.01),持续升高2小时后逐渐降低,12小时后基本恢复正常氧化还原状态。这与1μmol/L的AngⅡ作用于平滑肌细胞2h,应用非还原电泳检测到的ox-PTEN的表达逐渐升高的结果具有一致性。1μmol/L的AngⅡ使平滑肌细胞内受PTEN负调控的Akt及FAK通路被激活,p-Akt及p-FAK的表达均呈时间依赖性的升高,AngⅡ作用12小时后p-Akt的表达升高至正常对照组的4.5倍(p<0.001); p-FAK的表达升高至正常对照组的3.5倍(p<0.001)。三、血管紧张素Ⅱ影响平滑肌细胞内PTEN表达的机制研究预先用某些信号通路或因子的抑制剂、激动剂对平滑肌细胞进行预处理1小时,再加入1μmol/L的AngⅡ共同作用12小时,应用western blot和CCK-8法考察各种调节剂对细胞内PTEN表达水平的影响及对平滑肌细胞增殖的影响,以此分析AngⅡ影响PTEN表达水平的调控机制。western blot检测结果显示,AngⅡ受体抑制剂洛沙坦(losartan, Losa),过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor-y, PPARγ)激动剂罗格列酮(rosiglitazone, Rosi)及细胞核因子-κB (nuclear factor-KB, NF-κB)抑制剂BAY11-7082可以使PTEN的表达恢复甚至高于正常组PTEN的表达; c-jun氨基末端激酶(c-jun NH2-terminal kinase, JNK)抑制剂SP600125组PTEN的表达较AngⅡ组也略有提高；抗氧化剂α-硫辛酸(alpha-lipoic acid, a-LA)组细胞PTEN的表达与AngⅡ组相比无明显变化；而磷脂酰肌醇3激酶(phosphoinositide 3-kinase, PI3K)抑制剂LY294002,促分裂素原活化蛋白激酶(MAPK kinase 1/2 or ERK kinase 1/2, MEK1/2)抑制剂U0126及p38 MAPK抑制剂SB203580三组细胞PTEN的表达较AngⅡ组有不同程度的减少。细胞经相同的处理后,应用CCK-8法检测各种调节剂均对细胞的生长具有抑制作用。NF-κB抑制剂对细胞的生长抑制作用最为明显,抑制率达50%；P13K抑制剂,JNK抑制剂及p38 MAPK抑制剂对细胞生长的抑制率在20%左右；洛沙坦,罗格列酮及MEK1/2抑制剂对细胞生长的抑制率在10%左右；而α-硫辛酸(α-LA)对细胞生长的抑制率为4.23%,不具有统计学差异。本课题用AngⅡ作为诱因,证实在血管平滑肌细胞过度增殖和迁移的病理过程中抑癌基因PTEN活性和表达下降,因此受其负调控的下游Akt及FAK通路被激活,促使平滑肌细胞增殖和迁移。洛沙坦,罗格列酮以及NF-κB抑制剂可以逆转AngⅡ引起的PTEN表达的下降。抑癌基因PTEN可能成为治疗血管肥厚性疾病的重要靶点之一,具有深入研究的价值。更多还原

【Abstract】 Vascular smooth muscle cell (VSMC) is the only cellular component of vascular middle layer. Under physiological conditions, vascular smooth muscle regulates angiotasis by contraction and relaxation, and smooth muscle cells release some regulatory factors to maintain normal functions of blood vessels. Abnormal proliferation of VSMC, resulting in VSMC migration from media to intima, is critical event in the development of some vascular hypertrophy diseases such as coronary artery disease, percutaneous transluminal coronary angioplasty (PTCA) restenosis and vascular hypertrophy in hypertension. So finding the mechanisms and inhibiting abnormal proliferation of VSMC are important for the treatment of vascular hypertrophy diseases.Phosphatase and tensin homolog deleted on chromosome ten (PTEN), a new tumor suppressor, is a dual phosphatase tumor suppressor which expressed in central nervous system, heart, liver, kidney, gastrointestinal tract, lungs and skin as a constitutive secretion protein. PTEN involved in a variety of physiological processes, especially in cancer cells proliferation, differentiation, apoptosis, adhesion and migration. Recently, researchs about PTEN extended gradually from the cancer to some other fields such as cardiac hypertrophy, hypertension, atherosclerosis and bronchial asthma.Previous works have shown that as a dual phosphatase, PTEN can regulate phosphatidylinositol-3-kinase (PI3K), extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK) to inhibit tumor cell proliferation, migration and invasion. We suppose that PTEN have a similar effect on VSMC proliferation. In this study, we using AngⅡas stimulator to promote VSMC proliferation and migration, focused on the effects of PTEN and its pathways on AngⅡ-induced VSMC proliferation and migration and explored how to regulate the expression of PTEN.We confirmed AngⅡcould promote VSMC proliferation and migration, and identified the best concentration as well as effect time of AngⅡusing MTT, CCK-8 method and scratch experiments. The reslts of MTT assay confirmed 1μmol/L Ang II induced significant increase of VSMC proliferation. The proliferation rate was respectively 13.6% (p<0.001) after 12h and 13.9 (p<0.01) after 24h. Then CCK-8 assay had the similar results, proliferation rate was respectively 10.4% (p<0.01) after 12h and 9.8% (p<0.05) after 24h. Compared with the control cells, AngⅡcaused nearly 2 folds (P<0.05) increase of cell migration after 12h. Moreover, the expression of caspase-3 which can reflect the level of apoptosis was detected by western blot. The presence of AngⅡshowed to suppress the expression of caspase-3 in a time-dependent manner.To determine whether PTEN was involved in AngⅡ-mediated cell proliferation and migration, the expressions of PTEN, p-PTEN, re-PTEN, ox-PTEN after AngⅡstimulated were detected using western blot and nonreducing immunoblotting assay. And PTEN mRNA level was detected by RT-PCR. Intracellular free radicals were detected by DCFH-DA fluorescent probe. PTEN expression and PTEN mRNA decreased in response to AngⅡtreatment in a time-dependent manner. PTEN expression was reduced to only 1/4 compared with the control cells, and PTEN mRNA was decreased significantly after 2h of AngⅡtreatment. The level of p-PTEN was initially elevated after AngⅡstimulated 5min which was continued till 30min and then decreased gradually. Ox-PTEN level on the other hand was increased after exposure to AngⅡfrom 30min to 120min which was coincidence with intracellular reactive oxygen species (ROS) level. Collectively, these results indicate that AngⅡnot only decreased PTEN transcription and expression but also reduced PTEN activity by phosphorylation and oxidation PTEN protein. To assess whether the activity reduction of PTEN in response to AngⅡis linked to Akt and FAK signaling pathways, which are known to be involved in cell proliferation and migration, we examined the phosphorylation of Akt and FAK. The p-Akt and p-FAK levels were shown to be elevated initially from 30min and 5min respectively after AngⅡtreatment. And the expressions of p-Akt and p-FAK were shown to be increased as time dependent manner during 12h.Furthermore, VSMCs were pre-incubated with various pharmacological factors of signaling pathways for 1 h before AngⅡtreatment. Western blot and CCK-8 methods were used to examine PTEN expression and cell proliferation. The AT1 inhibitor losartan (20μmol/L), the PPARy agonist rosiglitazone (100μmol/L) and the NF-κB inhibitor BAY11-7082 (10μmol/L) significantly reversed the down-regulateion of PTEN expression induced by AngⅡ. And JNK inhibitor SP600125 (10μmol/L) could increased the expression of PTEN to some extent. All factors inhibited the proliferation of VSMC induced by AngⅡ. The growth suppression effect of NF-κB inhibitor BAY 11-7082 (10μmol/L), the PI3K inhibitors LY294002 (10μmol/L), the JNK inhibitor SP600125 (10μmol/L) and the p38 inhibitor SB203580 (10μmol/L) reached 20% or more.In the study, two major problems have been clarified. Firstly, the expression of tumor suppressor gene PTEN decreased over the pathological process of VSMC abnormal proliferation and migration was confirmed, using AngⅡas an incentive. Meanwhile, the expressions of inactive forms of PTEN, p-PTEN and ox-PTEN, increased rapidly but transiently after AngⅡstimulation. So that Akt and FAK pathways which are downstreams negative regulated by PTEN were activated. Secondly, results indicated losartan, rosiglitazone and NF-κB inhibitors could reverse down-regulation of PTEN expression caused by AngⅡ. PTEN may be an important target for the treatment of vascular hypertrophy diseases and is valuable to be in-depth studied and adequately understood.更多还原