本文選題：肝肺綜合征 + 肺動脈平滑肌細(xì)胞��； 參考：《第三軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：背景和目的： 肝肺綜合征(hepatopulmonary syndrome，HPS)主要發(fā)生在包括肝硬化在內(nèi)的終末期肝病患者中，其發(fā)生率超過15%，而死亡率逐年增加，因此對于HPS的研究逐漸成為一個熱點。肝源性細(xì)胞因子，導(dǎo)致肺內(nèi)微血管擴(kuò)張(intraplmonary vasodilatation,IPVD)，最終導(dǎo)致PASMCs的異常增殖，進(jìn)一步加重肺血管重建(pulmonary vascularremodelling，PVR)。因此，PASMCs的異常增殖是HPS相關(guān)PVR最主要的病理變化。我們前期的研究發(fā)現(xiàn)，HPS大鼠血清及3%濃度氧氣等可以誘導(dǎo)PASMCs異常增殖；同時，PASMCs發(fā)生表型轉(zhuǎn)化及其骨架蛋白也發(fā)生表達(dá)變化。既往研究發(fā)現(xiàn)，在一些疾病(如肺動脈高壓)的病理進(jìn)程中，PASMCs異常增殖的同時，伴隨著PASMCs的表型轉(zhuǎn)化和骨架重排；PASMCs的表型轉(zhuǎn)化及其骨架蛋白的表達(dá)變化是PASMCs發(fā)生異常增殖的上游途徑。尋找骨架蛋白變化和細(xì)胞增殖的關(guān)系，有利于進(jìn)一步解釋HPS的分子機(jī)制。 樁蛋白(Paxillin)是一種磷酸化蛋白，既往研究發(fā)現(xiàn)，其在多種細(xì)胞內(nèi)表達(dá)，參與多種細(xì)胞的生物學(xué)功能調(diào)節(jié)，在細(xì)胞骨架蛋白的表達(dá)與重排中起重要調(diào)控作用。既往研究發(fā)現(xiàn)，膜聯(lián)蛋白A1(annexin A1，ANXA1)可能和Paxillin具有相互作用，其可能影響Paxillin的表達(dá)，對Paxillin的研究是在前期研究上的深入，具有重要意義�；谏鲜鲆罁�(jù)，我們推測：在HPS相關(guān)的PASMCs異常增殖中，首先出現(xiàn)細(xì)胞骨架蛋白的表達(dá)變化和骨架重排以及細(xì)胞表型轉(zhuǎn)化，進(jìn)而導(dǎo)致PASMCs的異常增殖；Paxillin可能在細(xì)胞骨架蛋白的表達(dá)調(diào)節(jié)中起重要作用，并最終影響PASMCs的增殖。本研究擬通過膽總管結(jié)扎(common bile duct ligation，CBDL)構(gòu)建HPS大鼠模型，原代培養(yǎng)PASMCs，利用CCK-8、3H-TdR、RT-PCR及Western-blot等方法檢測PASMCs的增殖情況和其中Paxillin及骨架蛋白的表達(dá)情況；通過siRNA干擾技術(shù)對Paxillin進(jìn)行干預(yù)，探討其中的分子機(jī)制；最終揭示Paxillin在HPS相關(guān)PASMCs異常增殖中的分子機(jī)制，為HPS的預(yù)防和診治提供更好的措施。 方法： 實驗分為兩部分： (1)HPS大鼠血清培養(yǎng)的PASMCs中Paxillin及骨架蛋白表達(dá)的變化 1)構(gòu)建HPS大鼠模型及血清制備 2)大鼠PASMCs的分離、培養(yǎng)和鑒定 3)RT-PCR及Western-blot法檢測Paxillin的表達(dá)變化 4) Western-blot法檢測骨架蛋白(α-actin，，α-tubulin，destrin)的表達(dá)變化 (2)下調(diào)Paxillin的表達(dá)對HPS大鼠血清培養(yǎng)的PASMCs中骨架蛋白表達(dá)及其增殖的影響 1) siRNA下調(diào)PASMCs中Paxillin的表達(dá) 2)下調(diào)Paxillin表達(dá)后對HPS血清培養(yǎng)的PASMCs異常增殖及骨架蛋白表達(dá)的影響 結(jié)果： (1)HPS大鼠血清可以誘導(dǎo)Paxillin的轉(zhuǎn)錄和蛋白表達(dá)水平明顯上調(diào)，這一上調(diào)呈時間相關(guān)性。 (2)HPS大鼠血清刺激下PASMCs中骨架蛋白(α-actin，destrin，α-tubulin)表達(dá)呈時間相關(guān)性下調(diào)。 (3)利用特異性的Paxillin siRNA可以有效抑制Paxillin的蛋白表達(dá)。 (4)抑制Paxillin的表達(dá)后，HPS大鼠血清培養(yǎng)的PASMCs中骨架蛋白表達(dá)下調(diào)被抑制，PASMCs的異常增殖明顯受抑。 結(jié)論： 通過以上結(jié)果總結(jié)，在HPS相關(guān)的PASMCs異常增殖中，細(xì)胞骨架蛋白的表達(dá)變化可能是其異常增殖的前提；而在此過程中，Paxillin作為一個關(guān)鍵的調(diào)節(jié)靶點，其可能通過影響細(xì)胞骨架的表達(dá)變化調(diào)節(jié)PASMCs的增殖，即：Paxillin在HPS相關(guān)PASMCs增殖中具有重要調(diào)控作用。
[Abstract]:Background and purpose:
Hepatopulmonary syndrome (HPS) is mainly occurring in patients with end-stage liver disease including cirrhosis, the incidence of which is more than 15%, and the mortality rate is increasing year by year. Therefore, the study of HPS has become a hot spot. Hepatic cytokines, leading to the intraplmonary vasodilatation (IPVD), lead to the final guidance of the pulmonary microvascular expansion (IPVD). The abnormal proliferation of PASMCs further aggravates the pulmonary vascular reconstruction (pulmonary vascularremodelling, PVR). Therefore, the abnormal proliferation of PASMCs is the most important pathological change of HPS related PVR. Our previous study found that the serum and 3% concentration of oxygen in HPS rats can induce PASMCs ISO Chang Zengzhi; meanwhile, PASMCs has phenotypic transformation and its skeleton Previous studies have found that in the pathological process of some diseases (such as pulmonary arterial hypertension), the abnormal proliferation of PASMCs is accompanied by the phenotypic transformation of PASMCs and the skeleton rearrangement; the phenotypic transformation of PASMCs and the changes in the expression of the skeleton protein are the upstream pathways of the abnormal proliferation of PASMCs hair. The relationship between cell proliferation and cell proliferation is helpful to further explain the molecular mechanism of HPS.
Post protein (Paxillin) is a phosphorylated protein. Previous studies have found that it is expressed in a variety of cells and participates in the regulation of biological functions of various cells. It plays an important role in the expression and rearrangement of cytoskeleton protein. Previous studies have found that A1 (annexin A1, ANXA1) may interact with Paxillin, and its possible shadow On the basis of the above basis, we speculate that the changes in the expression of cytoskeleton protein, the rearrangement of cytoskeleton and the transformation of the cell phenotype, resulting in the abnormal proliferation of PASMCs in the HPS related PASMCs proliferation, and Paxillin may be possible, based on the above basis. The expression of cytoskeleton plays an important role in the regulation of the expression of cytoskeleton, and ultimately affects the proliferation of PASMCs. This study intends to construct a HPS rat model by common bile duct ligation (CBDL), the primary culture of PASMCs, and the use of CCK-8,3H-TdR, RT-PCR and Western-blot to detect the proliferation of PASMCs and its skeleton eggs. The expression of white; the molecular mechanism of Paxillin was discussed by siRNA interference technique, and the molecular mechanism of Paxillin in HPS related PASMCs abnormal proliferation was revealed, which provided better measures for the prevention and treatment of HPS.
Method:
The experiment is divided into two parts:
(1) changes of Paxillin and skeleton protein expression in PASMCs of HPS rats serum culture
1) the construction of HPS rat model and the preparation of serum
2) isolation, culture and identification of PASMCs in rats
3) the expression of Paxillin was detected by RT-PCR and Western-blot.
4) Western-blot method was used to detect the expression changes of -actin (-tubulin, destrin).
(2) down regulate the expression of Paxillin on the expression and proliferation of skeleton protein in PASMCs of HPS rats.
1) siRNA downregulation the expression of Paxillin in PASMCs
2) the effect of down regulating Paxillin expression on abnormal proliferation of PASMCs and expression of skeleton protein in HPS serum culture.
Result錛

本文編號：2056799