模拟失重下血管分化性改变的局部调节机制—L-RAS与收缩蛋白

发布时间：2018-06-17 18:44

本文选题：大鼠 + 模拟失重　；参考：《第四军医大学》2002年博士论文

【摘要】： 航天飞行后心血管失调发生机理及其对抗措施研究对阐明微重力心血管影响基本规律及实现新世纪载人航天目标具有重要的理论与实际意义。近年地面模拟研究及航天观察均表明，动脉血管的适应性变化在飞行后立位耐力不良发生中可能起重要作用。本实验室前期工作已发现，模拟失重可引起大鼠后身动脉血管发生萎缩性改变和收缩反应性降低，而脑部血管则发生肥厚和收缩反应性增强。这种分化性适应的调节机制目前尚不清楚，我们推想血管组织的局部肾素-血管紧张素系统(local renin-angiotensin system，L-RAS)可能发挥重要局部调控作用。故本工作应用RT-PCR和原位杂交、Western印迹分析等方法对模拟失重大鼠不同部位动脉血管血管紧张素原(angiotensinogen，AGT)及血管紧张素转化酶(angiotensin-converting enzyme，ACE)基因表达变化的时程特征进行了观察，并为后续工作制备了抗AGT多克隆抗血清。此外，我们的前期工作还提示，不同部位动脉血管平滑肌的收缩蛋白在模拟失重下也可能出现分化性变化，故本工作也用免疫组化及Westem印迹分析方法对模拟失重大鼠血管的α-平滑肌肌动蛋白(α-SM-actin)及调宁蛋白(calponin)表达变化进行了观察。第口旱区大学俗士学让茁穴本实验的主要发现如下： 1．心血管组织L－RAS主要成份的mRNA表达 RT．PCR分析结果表明，大鼠心肌、基底动脉、颈总动脉、腹主动脉和股动脉组织均有AGT和ACE的InRNA表达。原位杂交定位研究也进一步确认了AGT InRNA在心血管组织的表达：在左室心肌其弥散分布于心肌细胞；在血管壁不仅平滑肌层有较强的杂交信号，，在外膜甚至周围脂肪细胞也有表达。以往研究对心室肌是否表达L－RAS有较多争议，而对血管 LMS的研究则多限于主动脉，故本实验所发现的结果是对心血管组织可独立表达L．RAS主要成份这一问题的有力支持和重要补充。 2．4周模拟失重对不同部位动脉组织AGT和ACE mRNA表达的影咱半定量的RT．PCR分析结果表明，与同步对照大鼠相比，4周模拟失重大鼠基底动脉＊＊T的mRN A表达显著增加（P＜0刀5，n二5），而股动脉＊＊T mRNmRNA表达则呈减少趋势（P＞0刀5，。二5），在颈总动脉和腹主动脉则未见明显变化。在4周模拟失重大鼠上述血管组织的ACE mRNA表达与对照大鼠间无显著差别。本工作首次报道了模拟失重对不同部位动脉组织LRAS的分化性影响。 3．血管紧张素原多克隆抗血清的制备为研究AGT蛋白表达及后续工作，在大肠秆菌中融合表达了AGT蛋白的C－末端，以此融合蛋白为抗原免疫家兔，制备抗血清。经ELISA检测，其抗AGT效价可高达1：25600。Western印迹分析及免疫组化显示，所制备的抗血清不仅可识别原核表达的AGT片段，而且能够识别大鼠及人多种组织中内源性AGT蛋白。与己有抗体相比，本实验制备的抗AGT多克隆抗血清不仅具有较高的特异性，而且排除了与血管紧张素短片段的交叉反应。 4．不同部位血管组织AGT mRNA及蛋白表达的时程特征模拟失重大鼠基底动脉组织＊＊T m．nnfn．nnA表达水平随模拟时间延长而二二 7 第口旱匡大学博士学位沦文增高，2周组呈增加趋势，4周组达最高水平（P＜0刀5，n＝5），8周组仍维持在高的表达水平（＊＜0刀5，n＝5）：＊＊T蛋白表达水平呈相似的时程特征，4周组AGT含量最高，8周组仍维持在较高水平。而在股动脉组织，随模拟失重时间的延长，AGT rnRNA及蛋白的表达呈现逐渐降低的趋势，但与同步对照组间的差别均未达到显著水平。本发现表明，L－MS表达可能在模拟失重初期即己发生改变，并且始终维持在一定的水平，从而对不同部位动脉组织结构和功能的分化性适应发挥长时程的调控作用。 S．4周模拟失重对不同部位动脉组织a《M飞ctin及calponin表达的影响免疫组化方法显示，4周模拟失重大鼠基底动脉平滑肌细胞胞浆中 a．SM．actin阳性免疫反应颗粒较同步对照大鼠相比明显增多，在股动脉，阳性免疫反应颗粒则减少；Western印迹分析结果进一步验证了上述发现。而基底动脉和股动脉组织calponin表达量在模拟失重组与同步对照组大鼠之间未见明显改变。本实验所发现的
[Abstract]:The mechanism of cardiovascular dysfunction after space flight and its countermeasures are of great theoretical and practical significance to clarify the basic rules of microgravity cardiovascular influence and to realize the target of manned space flight in the new century. Early work in the laboratory has found that simulated weightlessness can cause atrophic and contractile responsiveness of the arteries of the posterior body of the rat, while the brain blood vessels have hypertrophy and contractile responsiveness. The regulatory mechanism of this differentiation is unclear. We think of local renin in vascular tissue. Local renin-angiotensin system (L-RAS) may play an important role in local regulation. Therefore, RT-PCR and in situ hybridization and Western blot analysis were used to simulate the arterial angiotensinogen (angiotensinogen, AGT) and angiotensin converting enzyme (angiotensin-converting) in different parts of the simulated weightless rats. Enzyme, ACE) the time history characteristics of gene expression changes were observed and the anti AGT polyclonal antiserum was prepared for the follow-up work. In addition, our earlier work also suggested that the contractile proteins of arterial smooth muscle in different parts of the artery may also be differentiated under simulated weightlessness, so this work also used immunohistochemical and Westem blotting analysis. Methods the expression of alpha smooth muscle actin (alpha -SM-actin) and calponin in vascular weightlessness rats were observed.
The University of Dryland
The main findings of this experiment are as follows:
1. mRNA expression of main components of cardiovascular tissue
RT.PCR analysis showed that the myocardium, basilar artery, common carotid artery and abdominal aorta in rats
The expression of AGT and ACE in InRNA and femoral artery tissues was also observed.
It confirmed the expression of AGT InRNA in cardiovascular tissue: the left ventricular myocardium distributed in the heart.
Muscle cells; in the vascular wall, not only the smooth muscle layer has strong hybridization signals, but also in the outer membrane or even surrounding fat.
There are also many controversies about whether Lv is expressed in ventricular myocardium in the past.
The study of LMS is mostly confined to aorta, so the result of this experiment is cardiovascular tissue.
Strong support and important supplement to the independent expression of the main components of L.RAS.
The effects of simulated weightlessness on the expression of AGT and ACE mRNA in different parts of arteries at 2.4 weeks
Semi quantitative RT.PCR analysis showed that the simulated weightlessness was 4 weeks compared with the synchronous control rats.
The expression of mRN A in T of basilar artery was significantly increased in rats (P < 0 knife 5, n two 5), while femoral artery
* the expression of T mRNmRNA showed a decreasing trend (P > 0 knife 5, two 5).
The expression of ACE mRNA in the above vascular tissues was simulated in weightlessness rats at 4 weeks.
There was no significant difference between the control group and the control group.
The effect of LRAS on the differentiation.
Preparation of 3. angiotensinogen polyclonal antiserum
In order to study the expression and subsequent work of AGT protein, AGT protein was expressed in E. coli.
The C-terminus was used to immunize rabbits with the fusion protein as antigen. The antiserum was prepared by ELISA.
Its anti AGT titers can be up to 1:25600.Western blot analysis and immunohistochemistry.
The antiserum not only recognizes prokaryotic expression of AGT fragments, but also identifies multiple groups of rats and humans.
The endogenous AGT protein in the weave. Compared with the existing antibody, the anti AGT polyclonal antiserum prepared in this experiment.
It not only has high specificity, but also excludes the cross reaction with short angiotensin.
4. the time history of AGT mRNA and protein expression in vascular tissues of different parts.
The expression level of T m.nnfn.nnA in basal artery tissue of simulated weightlessness rats prolonged with simulated time.
22
Seven
Doctoral degree in the University of Kuang Kuang
The increase in the 2 week group showed an increasing trend, and the highest level in 4 weeks (P < 0, 5, n = 5).
At a high level of expression (* < 0 knife 5, n = 5): * * T protein expression level is similar.
In the 4 week group, the AGT content was the highest, and the 8 week group remained at a higher level.
With the prolongation of simulated weightlessness, the expression of AGT rnRNA and protein decreased gradually.
However, the difference between the two groups was not significant. This finding indicates that the expression of L MS can be increased.
It can change at the early stage of simulated weightlessness, and it always stays at a certain level.
The adaptive differentiation of arterial tissue structure and function plays a long-term regulatory role.
Effects of simulated weightlessness on expression of aImmunohistochemical method showed that the cytoplasm of smooth muscle cells of basilar artery in 4 week simulated weightlessness rats
Compared with synchronous control rats, a.SM.actin positive immunoreactive granules increased significantly in femoral artery.
The positive immunoreactive granules decreased, and the results of Western blot analysis further verified the above findings.
The expression of calponin in basilar artery and femoral artery was simulated in the two groups.
There was no obvious change between rats.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2002
【分类号】：R852.22

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