本文選題：低切應力　切入點：RACK1　出處：《上海交通大學》2014年碩士論文　論文類型：學位論文

【摘要】：心血管疾病，如動脈粥樣硬化等給人類的生命健康帶來了重大的危害。各種病理因素導致的血管重建(Vascular remodeling)是這類疾病共有的發(fā)病基礎和基本的病理過程。 在體條件下的心血管系統(tǒng)，處于復雜的力學環(huán)境中，其中血流動力學是影響血管重建的重要因素。研究表明，，在血管分叉起始處和彎曲處容易形成動脈粥樣硬化，在這些區(qū)域主要存在的是低切應力（lowshear stress, LowSS），揭示LowSS在血管重建的發(fā)生和發(fā)展中有著重要的作用，然而其中的力學生物學機制還沒有被完全揭示。本實驗室前期發(fā)現(xiàn)，在體外應力培養(yǎng)的血管組織中，LowSS條件下，活化激酶C受體1（receptor for activated C kinase1, RACK1）的表達明顯升高，RACK1做為重要的蛋白激酶（protein kinase, PK）活性調控因子，其在切應力調控血管重建中的作用機制仍不明了。 基于此，本文對RACK1在血管細胞中的表達進行了驗證，并探討其在切應力調控血管內皮細胞（endothelial cells, ECs）和血管平滑肌細胞（vascular smooth muscle cells, VSMCs）增殖中的作用。本文運用聯(lián)合培養(yǎng)模型，培養(yǎng)大鼠胸主動脈ECs和VSMCs，應用平行平板流動腔系統(tǒng)，分別施加15dyn/cm2的正常切應力（normal shear stress,NSS）和5dyn/cm2的LowSS，加載時間為12h；然后用ELISA方法檢測細胞的增殖，應用Western blotting技術檢測RACK1表達及PKCα/βII、PKD、Akt磷酸化的變化。之后，在靜態(tài)條件下，運用RNA干擾方法，抑制ECs和VSMCs的RACK1表達，用BrdU ELISA方法檢測細胞的增殖，采用Western blotting技術檢測RACK1表達及PKCα/βII、PKD916、PKD744和Akt磷酸化的變化。 結果顯示：①與NSS相比，LowSS上調了ECs與VSMCs的增殖水平。②與NSS相比，在LowSS條件下，ECs與VSMCs中RACK1的表達水平，Akt、PKCα/βII的磷酸化顯著上調，同時LowSS顯著上調了PKD916位點絲氨酸殘基磷酸化，但對744位點絲氨酸殘基磷酸化無明顯作用。③靜態(tài)條件下，抑制ECs和VSMCs的RACK1表達引起細胞增殖下調。④靜態(tài)條件下，抑制ECs和VSMCs的RACK1表達，使PKC α/βII、PKD916、Akt的磷酸化水平下調。 結果表明，LowSS可誘導ECs和VSMCs的RACK1表達，從而活化PKCα/βII、PKD916、 Akt等多種細胞內信號分子，在血管重建時期促進細胞的增殖。
[Abstract]:Cardiovascular diseases, such as atherosclerosis, have brought great harm to human life and health. Vascular remodeling caused by various pathological factors is the common pathogenesis and basic pathological process of these diseases. In vivo, the cardiovascular system is in a complex mechanical environment, in which hemodynamics is an important factor affecting vascular remodeling. In these regions, low shear stress lowshear (LowSS) reveals that LowSS plays an important role in the occurrence and development of vascular remodeling, but the mechanism of mechanics and biology has not been fully revealed. Under the condition of LowSS, the expression of activated kinase C receptor for activated C kinase 1 (rack1) was significantly increased as an important regulatory factor of protein kinase protein kinase (PKK) activity. The mechanism of its role in the regulation of vascular remodeling by shear stress is still unclear. Based on this, the expression of RACK1 in vascular cells was verified, and the role of RACK1 in regulating the proliferation of vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMC) in vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMC) was investigated. ECs and VSMC were cultured in rat thoracic aorta. The normal shear stress shear (NSSs) of 15 dyn / cm 2 and low SS of 5 dyn / cm 2 were applied in parallel plate flow chamber system. The loading time was 12 h. Then the proliferation of cells was detected by ELISA method. Western blotting technique was used to detect the expression of RACK1 and the phosphorylation of PKC 偽 / 尾 PKC 偽 / 尾 PKDX. Then, under static condition, the RACK1 expression of ECs and VSMCs was inhibited by RNA interference method, and the proliferation of ECs and VSMCs was detected by BrdU ELISA method. The expression of RACK1 and the phosphorylation of PKC 偽 / 尾 -PKD916PKD744 and Akt were detected by Western blotting technique. The results showed that low SS upregulated the proliferation level of ECs and VSMCs. 2 compared with NSS, the expression level of RACK1 in LowSS and VSMCs was significantly up-regulated, and LowSS significantly up-regulated the phosphorylation of serine residues at PKD916 site. But there was no obvious effect on 744 site serine residue phosphorylation. Under static condition, inhibition of RACK1 expression of ECs and VSMCs resulted in down-regulation of cell proliferation under static condition, inhibition of RACK1 expression of ECs and VSMCs, and down-regulation of phosphorylation level of PKC 偽 / 尾 II-PKD916 / Akt. The results showed that low SS could induce the expression of RACK1 in ECs and VSMCs, thus activating many signal molecules such as PKC 偽 / 尾 II-PKD916, Akt and so on, which could promote the proliferation of cells during vascular reconstruction.
【學位授予單位】：上海交通大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R318.01

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相關期刊論文 前1條

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