Rab1介导的AT1R囊泡运输对低氧诱导的肺动脉平滑肌细胞表型转化及活性的影响

发布时间：2018-03-15 08:41

本文选题：肺动脉平滑肌细胞　切入点：表型转化　出处：《第三军医大学》2011年博士论文　论文类型：学位论文

【摘要】：背景和目的: 与处于终末期分化状态的骨骼肌细胞和心肌细胞不同,成年哺乳动物血管平滑肌细胞仍保持着高度的可塑性,当受到损伤时,可以发生可逆的表型转化。血管平滑肌细胞表型转化在人类多种疾病扮演着重要角色,如动脉粥样硬化、再狭窄、肺动脉高压和平滑肌肿瘤都与血管平滑肌细胞表型转化有着密切的关系。低氧可诱导肺动脉平滑肌细胞的表型转化,并且与多种信号分子有关,新近的研究表明血管紧张素II参与了低氧诱导的肺动脉平滑肌细胞增殖。血管紧张素II是一种辛肽化合物激素,通过与细胞膜表面特异的受体结合,进而激活多种细胞内信号传导途径,发挥生物学效应。血管紧张素II受体有两种亚型,血管紧张素II 1型受体和血管紧张素II 2型受体,二者均为七次跨膜G蛋白偶联受体超家族成员。血管紧张素II与血管紧张素II 1型受体结合可激活JAK/STAT途径和促分裂素原活化蛋白激酶(如细胞外调节激酶)。血管紧张素II 1型受体在血管重建和平滑肌细胞表型转化过程中具有重要意义,并且低氧可影响其在心肌细胞和血管平滑肌细胞中的分布。目前已有的研究表明,血管紧张素II 1型受体功能的实现与其在胞内的运输密切相关。在内质网,血管紧张素II 1型受体经过合成、折叠、装配后,输送到高尔基体进行翻译后的修饰,然后被输送到细胞膜表面。目前针对血管紧张素II 1型受体如何从内质网经过高尔基体到达细胞表面的运输过程的研究较少。 Rab蛋白,是GTPases的Ras超家族成员之一,与胞内蛋白在各细胞器之间的运输有关。Rab GTases被命名为小G蛋白,属于Ras超家族,已经发现有60多种Rab蛋白。Rab蛋白通过胞吐和胞吞的方式在囊泡运输中起重要作用,与其他Ras超家族成员一样,其功能通过与鸟嘌呤核苷酸结合时的分子转位实现。活性状态下(与GTP结合时),Rab蛋白能通过特定的效应器来介导囊泡运输。目前研究表明,Rab1分布于内质网和高尔基体,并介导内质网至高尔基体的顺向蛋白运输,Rab1可调节内皮细胞和心肌细胞血管紧张素II 1型受体从内质网经高尔基体到细胞表面的表达。因此我们设想,可以通过调整Rab1介导的血管紧张素II 1型受体在肺动脉平滑肌细胞内的运输,进而调节肺动脉平滑肌细胞的表型转化和其他活性。本实验主要研究低氧状态下,Rab1对肺动脉平滑肌细胞血管紧张素II 1型受体分布及功能的影响,并探讨Rab1对肺动脉平滑肌细胞表型转化和其他功能的影响,为改善肺血管重建提供新的治疗思路。研究内容: 1.采用肺内动脉组织贴块法培养大鼠肺动脉平滑肌细胞,通过光镜观察培养细胞特征性的形态,并使用特异性的α-SMA抗体进行免疫荧光染色鉴定。 2.运用饱和配体测定法测定大鼠肺动脉平滑肌细胞经低氧处理后、慢病毒包装的Rab1WT或siRNA转染后血管紧张素II 1型受体在胞膜上的表达。 3.使用激光共聚焦显微镜检测大鼠肺动脉平滑肌细胞转染慢病毒包装的Rab1WT和Rab1 siRNA后的血管紧张素II 1型受体在细胞膜和内质网的分布状况。 4.采用Western Blot检测大鼠肺动脉平滑肌细胞转染慢病毒包装的Rab1WT和Rab1 siRNA后的血管平滑肌细胞标志分子(α-SMA和VIM)的表达情况和STAT3活性。 5.细胞的增殖活性检测采用MTS分析法,细胞加入CellTiter 96? AQueous One Solution Cell Proliferation Assay后,使用酶标仪检测490nm的吸光度间接反映细胞的增殖活性。结果: 1.组织块法培养大鼠肺动脉平滑肌细胞的鉴定结果显示:相差显微镜下,原代培养的细胞呈梭状,并呈峰-谷分布生长,抗α-SMA抗体间接免疫荧光染色呈阳性。 2.正常对照组和低氧处理48 h后RPASMCs表面AT1R的表达量分别为556±61和725±83 cpm;常氧状态下,对照组、转染Rab1WT组和转染Rab siRNA组RPASMCs AT1R的表达值分别为550±69、804±130、301±46 cpm;低氧处理后,转染Rab1WT后的细胞AT1R的表达值738±98 cpm,转染Rab1 siRNA后的细胞AT1R的表达值仅为371±68 cpm(P 0.01)。 3.转染Rab1WT后,血管紧张素II 1型受体主要在肺动脉平滑肌细胞胞膜上表达,而转染Rab1 siRNA后肺动脉平滑肌细胞,血管紧张素II 1型受体主要积聚在内质网。 4.低氧处理以及使用血管紧张素II后,大鼠肺动脉平滑肌细胞STAT3酪氨酸磷酸化水平增加5.37倍,而转染Rab1 siRNA慢病毒后的细胞使用ZD7155(特异性的血管紧张素II 1型受体拮抗剂)后,其活性仅增加1.14倍(P0.01)。 5.低氧处理以及使用血管紧张素II后,大鼠肺动脉平滑肌细胞表型标志分子α-SMA和VIM含量明显减少,而转染Rab1 siRNA慢病毒后的细胞经ZD7155处理,其表型标志分子变化并不如此明显(P0.05)。 6.大鼠肺动脉平滑肌细胞经低氧处理以及使用血管紧张素II后,代表其增殖活性的OD值较常氧对照组增加159%,而转染Rab1 siRNA慢病毒后的细胞并使用ZD7155处理后,其OD值仅增加27%(P0.01)。结论: 1. Rab1蛋白可调节AT1R在大鼠肺动脉平滑肌细胞的分布,其机制可能是通过影响AT1R在胞内的囊泡运输。 2. Rab1蛋白可调节AT1R介导的JAK/STAT信号转导途径。 3. Rab1蛋白通过调节AT1R在大鼠肺动脉平滑肌细胞的分布,参与低氧诱导的大鼠肺动脉平滑肌细胞由分化型向去分化型的转化。 4. Rab1蛋白参与调节大鼠肺动脉平滑肌细胞的增殖活性。综上所述,Rab1可以作为改善肺血管重建的潜在治疗靶点。
[Abstract]:Background and purpose:
Unlike in end-stage differentiation of skeletal muscle cells and myocardial cells, vascular smooth muscle cells of the adult mammalian still maintains a high degree of plasticity, when damaged, the transformed phenotype can occur reversible. The phenotype of vascular smooth muscle cell transformation plays an important role in many human diseases such as atherosclerosis, restenosis, and pulmonary arterial hypertension smooth muscle tumors are associated with phenotypic modulation in vascular smooth muscle cells have a close relationship. The transformed phenotype can be induced by hypoxia in pulmonary artery smooth muscle cells, and is associated with a variety of signaling molecules, recent studies suggest that angiotensin II is involved in the proliferation of pulmonary artery smooth muscle cells induced by hypoxia.
Angiotensin II is an octapeptide hormone, with specific cell surface receptors, then activates multiple intracellular signaling pathways play a biological effect. Angiotensin II receptor has two isoforms, angiotensin II type 1 receptor and angiotensin II type 2 receptor, two are seven transmembrane G protein coupled receptor superfamily. Angiotensin II and angiotensin II type 1 receptor can activate JAK/STAT pathway and mitogen activated protein kinase (such as extracellular regulated kinase). It is very important to angiotensin II type 1 receptor phenotype of smooth muscle cells in tube reconstruction the blood in the process of transformation, and hypoxia can influence its distribution in myocardial cells and vascular smooth muscle cells.
The present study shows that implementation of angiotensin II type 1 receptor and its function in intracellular transport is closely related. In the endoplasmic reticulum, angiotensin II type 1 receptor after synthesis, folding, assembly, transport to the Golgi apparatus was modified after translation, then transported to the cell membrane surface. At present angiotensin II type 1 receptor from the endoplasmic reticulum through the Golgi transport process to study the cell surface is less.
Rab protein, GTPases Ras superfamily, and intracellular protein trafficking between organelles.Rab GTases called G protein, belonging to the Ras superfamily, has been found in 60 kinds of Rab.Rab protein by exocytosis and endocytosis in the vesicles play an important role in the transportation. As with other members of the Ras superfamily, and its function by guanine nucleotide binding molecules. The transposition of the active state (combined with the GTP), Rab protein by specific effectors mediated vesicular transport. The present study showed that the distribution of Rab1 in the endoplasmic reticulum and Golgi, mediated endoplasmic reticulum to the Golgi tendem protein transport, Rab1 regulates endothelial cells and myocardial cells of angiotensin II type 1 receptor expression from the endoplasmic reticulum to the cell surface by Golgi. So we can imagine, angiotensin II by adjusting the Rab1 mediated blood tube The type 1 receptor is transported in the pulmonary artery smooth muscle cells, and then regulates the phenotypic transformation and other activity of the pulmonary artery smooth muscle cells.
In this study, we studied the effects of Rab1 on the distribution and function of angiotensin II 1 receptor in pulmonary artery smooth muscle cells under hypoxia, and explored the effects of Rab1 on phenotypic transformation and other functions of pulmonary artery smooth muscle cells, so as to provide new ideas for improving pulmonary vascular remodeling.
Research content:
1., rat pulmonary artery smooth muscle cells were cultured by intrapulmonary artery tissue block method. The morphological characteristics of cultured cells were observed by light microscope, and the specific -SMA antibody was used to identify the immunofluorescent staining.
2. the expression of ang 1 II receptor on the membrane of rat pulmonary artery smooth muscle cells after hypoxia treatment was detected by saturated ligand assay after transfection of lentivirus packaged Rab1WT or siRNA.
3., we used laser confocal microscopy to detect the distribution of angiotensin II 1 receptor in cell membrane and endoplasmic reticulum in rat pulmonary artery smooth muscle cells transfected with lentivirus packaged Rab1WT and Rab1 siRNA.
4. Western Blot was used to detect the expression and STAT3 activity of vascular smooth muscle cells markers (alpha -SMA and VIM) in rat pulmonary artery smooth muscle cells transfected with lentivirus packaged Rab1WT and Rab1 siRNA.
5., the proliferation activity of cells was detected by MTS analysis. After adding CellTiter 96, AQueous One Solution Cell Proliferation Assay, the absorbance of 490nm was detected by enzyme labelling, which indirectly reflected cell proliferative activity.
Result:
1. identification of rat pulmonary artery smooth muscle cells by tissue explant method showed that the primary cultured cells were spindle shaped and showed peak valley distribution under phase contrast microscope, and the anti -SMA antibody was positive by indirect immunofluorescence staining.
2. normal control group and hypoxia 48 h surface expression of RPASMCs AT1R were 556 + 61 and 725 + 83 CPM; under normoxic condition, control group, Rab1WT group and Rab transfection and expression of transfected siRNA group RPASMCs AT1R = 550 + 69804 + 130301 + 46 CPM; after hypoxia, the expression of after transfection of Rab1WT cells with AT1R values of 738 + 98 CPM, the value of siRNA cells after transfection and expression of Rab1 AT1R was 371 + 68 CPM (P 0.01).
3. after transfection of Rab1WT, the angiotensin II 1 receptor was mainly expressed on the pulmonary artery smooth muscle cell membrane. After transfection of Rab1 siRNA, the angiotensin II 1 receptor was mainly accumulated in the endoplasmic reticulum.
4. hypoxia treatment and use of angiotensin II, rat pulmonary artery smooth muscle cells STAT3 tyrosine phosphorylation increased 5.37 times, while the siRNA lentivirus transfected Rab1 cells after using ZD7155 (specific angiotensin II type 1 receptor antagonist), its activity increased only 1.14 times (P0.01).
5., after hypoxic treatment and angiotensin II, the phenotypic markers, -SMA and VIM contents of rat pulmonary artery smooth muscle cells were significantly reduced. However, after ZD7155 treatment, the phenotypic markers of Rab1 siRNA lentivirus cells did not change significantly (P0.05).
6. rat pulmonary artery smooth muscle cells after hypoxia treatment and the use of angiotensin II, OD on behalf of its proliferation activity compared with that in the normoxia group increased 159%, while Rab1 siRNA lentivirus transfected cells after and after ZD7155 treatment, the OD value increased by only 27% (P0.01).
Conclusion:
1. Rab1 protein regulates the distribution of AT1R in the rat pulmonary artery smooth muscle cells, which may be mediated by the effect of the transport of AT1R in the cytosolic vesicles.
2. Rab1 protein can regulate the AT1R mediated JAK/STAT signal transduction pathway.
3. Rab1 protein regulates the distribution of AT1R in rat pulmonary artery smooth muscle cells and participates in the transformation of rat pulmonary artery smooth muscle cells from differentiated to dedifferentiated.
4. Rab1 protein is involved in regulating the proliferation activity of rat pulmonary artery smooth muscle cells.
To sum up, Rab1 can be used as a potential therapeutic target for the improvement of pulmonary vascular reconstruction.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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