Paxillin在HPS相关PASMCs异常增殖中的作用和机制研究

发布时间：2018-06-23 10:21

  本文选题：肝肺综合征 + 肺动脉平滑肌细胞　； 参考：《第三军医大学》2014年硕士论文

【摘要】：背景和目的： 肝肺综合征(hepatopulmonary syndrome，HPS)主要发生在包括肝硬化在内的终末期肝病患者中，其发生率超过15%，而死亡率逐年增加，因此对于HPS的研究逐渐成为一个热点。肝源性细胞因子，导致肺内微血管扩张(intraplmonary vasodilatation,IPVD)，最终导致PASMCs的异常增殖，进一步加重肺血管重建(pulmonary vascularremodelling，PVR)。因此，PASMCs的异常增殖是HPS相关PVR最主要的病理变化。我们前期的研究发现，HPS大鼠血清及3%浓度氧气等可以诱导PASMCs异常增殖；同时，PASMCs发生表型转化及其骨架蛋白也发生表达变化。既往研究发现，在一些疾病(如肺动脉高压)的病理进程中，PASMCs异常增殖的同时，伴随着PASMCs的表型转化和骨架重排；PASMCs的表型转化及其骨架蛋白的表达变化是PASMCs发生异常增殖的上游途径。寻找骨架蛋白变化和细胞增殖的关系，有利于进一步解释HPS的分子机制。 桩蛋白(Paxillin)是一种磷酸化蛋白，既往研究发现，其在多种细胞内表达，参与多种细胞的生物学功能调节，在细胞骨架蛋白的表达与重排中起重要调控作用。既往研究发现，膜联蛋白A1(annexin A1，ANXA1)可能和Paxillin具有相互作用，其可能影响Paxillin的表达，对Paxillin的研究是在前期研究上的深入，具有重要意义。基于上述依据，我们推测：在HPS相关的PASMCs异常增殖中，首先出现细胞骨架蛋白的表达变化和骨架重排以及细胞表型转化，进而导致PASMCs的异常增殖；Paxillin可能在细胞骨架蛋白的表达调节中起重要作用，并最终影响PASMCs的增殖。本研究拟通过胆总管结扎(common bile duct ligation，CBDL)构建HPS大鼠模型，原代培养PASMCs，利用CCK-8、3H-TdR、RT-PCR及Western-blot等方法检测PASMCs的增殖情况和其中Paxillin及骨架蛋白的表达情况；通过siRNA干扰技术对Paxillin进行干预，探讨其中的分子机制；最终揭示Paxillin在HPS相关PASMCs异常增殖中的分子机制，为HPS的预防和诊治提供更好的措施。 方法： 实验分为两部分： (1)HPS大鼠血清培养的PASMCs中Paxillin及骨架蛋白表达的变化 1)构建HPS大鼠模型及血清制备 2)大鼠PASMCs的分离、培养和鉴定 3)RT-PCR及Western-blot法检测Paxillin的表达变化 4) Western-blot法检测骨架蛋白(α-actin，，α-tubulin，destrin)的表达变化 (2)下调Paxillin的表达对HPS大鼠血清培养的PASMCs中骨架蛋白表达及其增殖的影响 1) siRNA下调PASMCs中Paxillin的表达 2)下调Paxillin表达后对HPS血清培养的PASMCs异常增殖及骨架蛋白表达的影响 结果： (1)HPS大鼠血清可以诱导Paxillin的转录和蛋白表达水平明显上调，这一上调呈时间相关性。 (2)HPS大鼠血清刺激下PASMCs中骨架蛋白(α-actin，destrin，α-tubulin)表达呈时间相关性下调。 (3)利用特异性的Paxillin siRNA可以有效抑制Paxillin的蛋白表达。 (4)抑制Paxillin的表达后，HPS大鼠血清培养的PASMCs中骨架蛋白表达下调被抑制，PASMCs的异常增殖明显受抑。 结论： 通过以上结果总结，在HPS相关的PASMCs异常增殖中，细胞骨架蛋白的表达变化可能是其异常增殖的前提；而在此过程中，Paxillin作为一个关键的调节靶点，其可能通过影响细胞骨架的表达变化调节PASMCs的增殖，即：Paxillin在HPS相关PASMCs增殖中具有重要调控作用。
[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锛

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