本文選題：大鼠 + 模擬失重��； 參考：《第四軍醫(yī)大學(xué)》2002年博士論文

【摘要】： 航天飛行后心血管失調(diào)發(fā)生機(jī)理及其對抗措施研究對闡明微重力心血管影響基本規(guī)律及實現(xiàn)新世紀(jì)載人航天目標(biāo)具有重要的理論與實際意義。近年地面模擬研究及航天觀察均表明，動脈血管的適應(yīng)性變化在飛行后立位耐力不良發(fā)生中可能起重要作用。本實驗室前期工作已發(fā)現(xiàn)，模擬失重可引起大鼠后身動脈血管發(fā)生萎縮性改變和收縮反應(yīng)性降低，而腦部血管則發(fā)生肥厚和收縮反應(yīng)性增強(qiáng)。這種分化性適應(yīng)的調(diào)節(jié)機(jī)制目前尚不清楚，我們推想血管組織的局部腎素-血管緊張素系統(tǒng)(local renin-angiotensin system，L-RAS)可能發(fā)揮重要局部調(diào)控作用。故本工作應(yīng)用RT-PCR和原位雜交、Western印跡分析等方法對模擬失重大鼠不同部位動脈血管血管緊張素原(angiotensinogen，AGT)及血管緊張素轉(zhuǎn)化酶(angiotensin-converting enzyme，ACE)基因表達(dá)變化的時程特征進(jìn)行了觀察，并為后續(xù)工作制備了抗AGT多克隆抗血清。此外，我們的前期工作還提示，不同部位動脈血管平滑肌的收縮蛋白在模擬失重下也可能出現(xiàn)分化性變化，故本工作也用免疫組化及Westem印跡分析方法對模擬失重大鼠血管的α-平滑肌肌動蛋白(α-SM-actin)及調(diào)寧蛋白(calponin)表達(dá)變化進(jìn)行了觀察。 第口旱區(qū)大學(xué)俗士學(xué)讓茁穴 本實驗的主要發(fā)現(xiàn)如下： 1．心血管組織L－RAS主要成份的mRNA表達(dá) RT．PCR分析結(jié)果表明，大鼠心肌、基底動脈、頸總動脈、腹主動脈 和股動脈組織均有AGT和ACE的InRNA表達(dá)。原位雜交定位研究也進(jìn)一 步確認(rèn)了AGT InRNA在心血管組織的表達(dá)：在左室心肌其彌散分布于心 肌細(xì)胞；在血管壁不僅平滑肌層有較強(qiáng)的雜交信號，，在外膜甚至周圍脂肪 細(xì)胞也有表達(dá)。以往研究對心室肌是否表達(dá)L－RAS有較多爭議，而對血管 LMS的研究則多限于主動脈，故本實驗所發(fā)現(xiàn)的結(jié)果是對心血管組織可 獨立表達(dá)L．RAS主要成份這一問題的有力支持和重要補(bǔ)充。 2．4周模擬失重對不同部位動脈組織AGT和ACE mRNA表達(dá)的影咱 半定量的RT．PCR分析結(jié)果表明，與同步對照大鼠相比，4周模擬失重 大鼠基底動脈＊＊T的mRN A表達(dá)顯著增加（P＜0刀5，n二5），而股動脈 ＊＊T mRNmRNA表達(dá)則呈減少趨勢（P＞0刀5，。二5），在頸總動脈和腹主動 脈則未見明顯變化。在4周模擬失重大鼠上述血管組織的ACE mRNA表達(dá) 與對照大鼠間無顯著差別。本工作首次報道了模擬失重對不同部位動脈組 織LRAS的分化性影響。 3．血管緊張素原多克隆抗血清的制備 為研究AGT蛋白表達(dá)及后續(xù)工作，在大腸稈菌中融合表達(dá)了AGT蛋白 的C－末端，以此融合蛋白為抗原免疫家兔，制備抗血清。經(jīng)ELISA檢測， 其抗AGT效價可高達(dá)1：25600。Western印跡分析及免疫組化顯示，所制備 的抗血清不僅可識別原核表達(dá)的AGT片段，而且能夠識別大鼠及人多種組 織中內(nèi)源性AGT蛋白。與己有抗體相比，本實驗制備的抗AGT多克隆抗血 清不僅具有較高的特異性，而且排除了與血管緊張素短片段的交叉反應(yīng)。 4．不同部位血管組織AGT mRNA及蛋白表達(dá)的時程特征 模擬失重大鼠基底動脈組織＊＊T m．nnfn．nnA表達(dá)水平隨模擬時間延長而 二二 7 第口旱匡大學(xué)博士學(xué)位淪文 增高，2周組呈增加趨勢，4周組達(dá)最高水平（P＜0刀5，n＝5），8周組仍 維持在高的表達(dá)水平（＊＜0刀5，n＝5）：＊＊T蛋白表達(dá)水平呈相似的時 程特征，4周組AGT含量最高，8周組仍維持在較高水平。而在股動脈組織， 隨模擬失重時間的延長，AGT rnRNA及蛋白的表達(dá)呈現(xiàn)逐漸降低的趨勢， 但與同步對照組間的差別均未達(dá)到顯著水平。本發(fā)現(xiàn)表明，L－MS表達(dá)可 能在模擬失重初期即己發(fā)生改變，并且始終維持在一定的水平，從而對不 同部位動脈組織結(jié)構(gòu)和功能的分化性適應(yīng)發(fā)揮長時程的調(diào)控作用。 S．4周模擬失重對不同部位動脈組織a《M飛ctin及calponin表達(dá)的影響 免疫組化方法顯示，4周模擬失重大鼠基底動脈平滑肌細(xì)胞胞漿中 a．SM．a(chǎn)ctin陽性免疫反應(yīng)顆粒較同步對照大鼠相比明顯增多，在股動脈， 陽性免疫反應(yīng)顆粒則減少；Western印跡分析結(jié)果進(jìn)一步驗證了上述發(fā) 現(xiàn)。而基底動脈和股動脈組織calponin表達(dá)量在模擬失重組與同步對照組 大鼠之間未見明顯改變。本實驗所發(fā)現(xiàn)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2002
【分類號】：R852.22

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