本文選題：肺動脈平滑肌細胞　切入點：表型轉(zhuǎn)化　出處：《第三軍醫(yī)大學(xué)》2011年博士論文　論文類型：學(xué)位論文

【摘要】：背景和目的: 與處于終末期分化狀態(tài)的骨骼肌細胞和心肌細胞不同,成年哺乳動物血管平滑肌細胞仍保持著高度的可塑性,當(dāng)受到損傷時,可以發(fā)生可逆的表型轉(zhuǎn)化。血管平滑肌細胞表型轉(zhuǎn)化在人類多種疾病扮演著重要角色,如動脈粥樣硬化、再狹窄、肺動脈高壓和平滑肌腫瘤都與血管平滑肌細胞表型轉(zhuǎn)化有著密切的關(guān)系。低氧可誘導(dǎo)肺動脈平滑肌細胞的表型轉(zhuǎn)化,并且與多種信號分子有關(guān),新近的研究表明血管緊張素II參與了低氧誘導(dǎo)的肺動脈平滑肌細胞增殖。 血管緊張素II是一種辛肽化合物激素,通過與細胞膜表面特異的受體結(jié)合,進而激活多種細胞內(nèi)信號傳導(dǎo)途徑,發(fā)揮生物學(xué)效應(yīng)。血管緊張素II受體有兩種亞型,血管緊張素II 1型受體和血管緊張素II 2型受體,二者均為七次跨膜G蛋白偶聯(lián)受體超家族成員。血管緊張素II與血管緊張素II 1型受體結(jié)合可激活JAK/STAT途徑和促分裂素原活化蛋白激酶(如細胞外調(diào)節(jié)激酶)。血管緊張素II 1型受體在血管重建和平滑肌細胞表型轉(zhuǎn)化過程中具有重要意義,并且低氧可影響其在心肌細胞和血管平滑肌細胞中的分布。 目前已有的研究表明,血管緊張素II 1型受體功能的實現(xiàn)與其在胞內(nèi)的運輸密切相關(guān)。在內(nèi)質(zhì)網(wǎng),血管緊張素II 1型受體經(jīng)過合成、折疊、裝配后,輸送到高爾基體進行翻譯后的修飾,然后被輸送到細胞膜表面。目前針對血管緊張素II 1型受體如何從內(nèi)質(zhì)網(wǎng)經(jīng)過高爾基體到達細胞表面的運輸過程的研究較少。 Rab蛋白,是GTPases的Ras超家族成員之一,與胞內(nèi)蛋白在各細胞器之間的運輸有關(guān)。Rab GTases被命名為小G蛋白,屬于Ras超家族,已經(jīng)發(fā)現(xiàn)有60多種Rab蛋白。Rab蛋白通過胞吐和胞吞的方式在囊泡運輸中起重要作用,與其他Ras超家族成員一樣,其功能通過與鳥嘌呤核苷酸結(jié)合時的分子轉(zhuǎn)位實現(xiàn)�；钚誀顟B(tài)下(與GTP結(jié)合時),Rab蛋白能通過特定的效應(yīng)器來介導(dǎo)囊泡運輸。目前研究表明,Rab1分布于內(nèi)質(zhì)網(wǎng)和高爾基體,并介導(dǎo)內(nèi)質(zhì)網(wǎng)至高爾基體的順向蛋白運輸,Rab1可調(diào)節(jié)內(nèi)皮細胞和心肌細胞血管緊張素II 1型受體從內(nèi)質(zhì)網(wǎng)經(jīng)高爾基體到細胞表面的表達。因此我們設(shè)想,可以通過調(diào)整Rab1介導(dǎo)的血管緊張素II 1型受體在肺動脈平滑肌細胞內(nèi)的運輸,進而調(diào)節(jié)肺動脈平滑肌細胞的表型轉(zhuǎn)化和其他活性。 本實驗主要研究低氧狀態(tài)下,Rab1對肺動脈平滑肌細胞血管緊張素II 1型受體分布及功能的影響,并探討Rab1對肺動脈平滑肌細胞表型轉(zhuǎn)化和其他功能的影響,為改善肺血管重建提供新的治療思路。 研究內(nèi)容: 1.采用肺內(nèi)動脈組織貼塊法培養(yǎng)大鼠肺動脈平滑肌細胞,通過光鏡觀察培養(yǎng)細胞特征性的形態(tài),并使用特異性的α-SMA抗體進行免疫熒光染色鑒定。 2.運用飽和配體測定法測定大鼠肺動脈平滑肌細胞經(jīng)低氧處理后、慢病毒包裝的Rab1WT或siRNA轉(zhuǎn)染后血管緊張素II 1型受體在胞膜上的表達。 3.使用激光共聚焦顯微鏡檢測大鼠肺動脈平滑肌細胞轉(zhuǎn)染慢病毒包裝的Rab1WT和Rab1 siRNA后的血管緊張素II 1型受體在細胞膜和內(nèi)質(zhì)網(wǎng)的分布狀況。 4.采用Western Blot檢測大鼠肺動脈平滑肌細胞轉(zhuǎn)染慢病毒包裝的Rab1WT和Rab1 siRNA后的血管平滑肌細胞標志分子(α-SMA和VIM)的表達情況和STAT3活性。 5.細胞的增殖活性檢測采用MTS分析法,細胞加入CellTiter 96? AQueous One Solution Cell Proliferation Assay后,使用酶標儀檢測490nm的吸光度間接反映細胞的增殖活性。 結(jié)果: 1.組織塊法培養(yǎng)大鼠肺動脈平滑肌細胞的鑒定結(jié)果顯示:相差顯微鏡下,原代培養(yǎng)的細胞呈梭狀,并呈峰-谷分布生長,抗α-SMA抗體間接免疫熒光染色呈陽性。 2.正常對照組和低氧處理48 h后RPASMCs表面AT1R的表達量分別為556±61和725±83 cpm;常氧狀態(tài)下,對照組、轉(zhuǎn)染Rab1WT組和轉(zhuǎn)染Rab siRNA組RPASMCs AT1R的表達值分別為550±69、804±130、301±46 cpm;低氧處理后,轉(zhuǎn)染Rab1WT后的細胞AT1R的表達值738±98 cpm,轉(zhuǎn)染Rab1 siRNA后的細胞AT1R的表達值僅為371±68 cpm(P 0.01)。 3.轉(zhuǎn)染Rab1WT后,血管緊張素II 1型受體主要在肺動脈平滑肌細胞胞膜上表達,而轉(zhuǎn)染Rab1 siRNA后肺動脈平滑肌細胞,血管緊張素II 1型受體主要積聚在內(nèi)質(zhì)網(wǎng)。 4.低氧處理以及使用血管緊張素II后,大鼠肺動脈平滑肌細胞STAT3酪氨酸磷酸化水平增加5.37倍,而轉(zhuǎn)染Rab1 siRNA慢病毒后的細胞使用ZD7155(特異性的血管緊張素II 1型受體拮抗劑)后,其活性僅增加1.14倍(P0.01)。 5.低氧處理以及使用血管緊張素II后,大鼠肺動脈平滑肌細胞表型標志分子α-SMA和VIM含量明顯減少,而轉(zhuǎn)染Rab1 siRNA慢病毒后的細胞經(jīng)ZD7155處理,其表型標志分子變化并不如此明顯(P0.05)。 6.大鼠肺動脈平滑肌細胞經(jīng)低氧處理以及使用血管緊張素II后,代表其增殖活性的OD值較常氧對照組增加159%,而轉(zhuǎn)染Rab1 siRNA慢病毒后的細胞并使用ZD7155處理后,其OD值僅增加27%(P0.01)。 結(jié)論: 1. Rab1蛋白可調(diào)節(jié)AT1R在大鼠肺動脈平滑肌細胞的分布,其機制可能是通過影響AT1R在胞內(nèi)的囊泡運輸。 2. Rab1蛋白可調(diào)節(jié)AT1R介導(dǎo)的JAK/STAT信號轉(zhuǎn)導(dǎo)途徑。 3. Rab1蛋白通過調(diào)節(jié)AT1R在大鼠肺動脈平滑肌細胞的分布,參與低氧誘導(dǎo)的大鼠肺動脈平滑肌細胞由分化型向去分化型的轉(zhuǎn)化。 4. Rab1蛋白參與調(diào)節(jié)大鼠肺動脈平滑肌細胞的增殖活性。 綜上所述,Rab1可以作為改善肺血管重建的潛在治療靶點。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R363

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