發(fā)布時(shí)間：2018-03-25 15:18

  本文選題：大鼠　切入點(diǎn)：失重模擬　出處：《中國人民解放軍軍醫(yī)進(jìn)修學(xué)院》2009年博士論文

【摘要】： 目的: 失重會使肺功能受到影響,并可導(dǎo)致一定程度肺組織損傷。本實(shí)驗(yàn)采用大鼠尾懸吊模擬失重的方法,研究模擬失重后大鼠肺組織蛋白組學(xué)變化情況,深入了解失重所致肺損傷的發(fā)生機(jī)制;同時(shí)選擇N-乙酰半胱氨酸和銀杏葉提取物進(jìn)行藥物干預(yù),觀察大鼠肺組織細(xì)胞凋亡和一氧化氮表達(dá)情況的變化,為失重所致肺損傷的藥物防護(hù)提供理論依據(jù)。 方法: 1.蛋白組學(xué)研究:選擇健康雄性S-D大鼠20只,體重180-220g,隨機(jī)分為模擬失重7天和正常對照2組,采用尾懸吊-30°作為模擬失重模型。尾懸吊結(jié)束時(shí),用10%水合氯醛(30ml/kg)腹腔注射麻醉后,打開胸腔取出肺組織并提取肺組織蛋白,蛋白提取后采用雙向電泳技術(shù)(2-DE)建立差異表達(dá)圖譜,通過凝膠成像分析和人工比對,確定差異蛋白質(zhì)斑點(diǎn),然后對每個(gè)差異蛋白點(diǎn)進(jìn)行膠內(nèi)酶切,采用HDMS進(jìn)行液相串聯(lián)質(zhì)譜(LC-MS/MS)分析結(jié)合數(shù)據(jù)庫檢索鑒定蛋白質(zhì)。最后對差異蛋白的生物學(xué)功能進(jìn)行較為詳細(xì)的了解和分類,進(jìn)一步深入研究它們的功能。 2.藥物干預(yù)研究:選擇健康雄性S-D大鼠40只,體重180-220g,隨機(jī)分為模擬失重7天、正常對照、N-乙酰半胱氨酸干預(yù)和銀杏葉提取物干預(yù)4組,采用尾懸吊-30°作為模擬失重模型。尾懸吊結(jié)束時(shí),用10%水合氯醛(30ml/kg)腹腔注射麻醉后,打開胸腔取出肺組織制作石蠟切片。采用原位細(xì)胞凋亡檢測法、免疫組織化學(xué)法和原位雜交法分別觀察大鼠肺組織細(xì)胞凋亡、誘導(dǎo)型一氧化氮合酶(iNOS)和bcl-2、bax及iNOS mRNA的變化。 結(jié)果: 1.蛋白組學(xué)變化:應(yīng)用2-DE對懸吊組和對照組大鼠肺組織樣品進(jìn)行分離和對比,建立差異表達(dá)圖譜,通過凝膠成像分析和人工比對,確定差異蛋白質(zhì)斑點(diǎn)17個(gè),其中13個(gè)上調(diào),3個(gè)下調(diào),1個(gè)消失。這些點(diǎn)分布在等電點(diǎn)4.65-8.61、分子量15972-60317范圍內(nèi),最大表達(dá)上調(diào)6.9倍,下調(diào)最低至0.1倍。經(jīng)膠內(nèi)酶切和質(zhì)譜鑒定,共鑒定出17種蛋白質(zhì),其中13種上調(diào),3種下調(diào),1種消失,與細(xì)胞代謝相關(guān)的5個(gè),與細(xì)胞功能相關(guān)的4個(gè),與細(xì)胞結(jié)構(gòu)蛋白相關(guān)的3個(gè),與蛋白降解系統(tǒng)相關(guān)的2個(gè),與信號傳導(dǎo)相關(guān)的3個(gè)。它們可能與細(xì)胞能量代謝、應(yīng)激與炎癥反應(yīng)、細(xì)胞損傷與修復(fù)、細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)和其它細(xì)胞功能活動有關(guān),在細(xì)胞免疫應(yīng)答、細(xì)胞凋亡等方面起著重要的作用。 2.藥物干預(yù)結(jié)果:懸吊組大鼠肺組織細(xì)胞凋亡指數(shù)(23.1%±2.6%)明顯高于正常對照組(2.2%±1.1%)(P＜0.05),而N-乙酰半胱氨酸干預(yù)組大鼠肺組織細(xì)胞凋亡指數(shù)(7.5%±1.6%)顯著低于尾懸吊組(P＜0.05),大鼠肺組織bcl-2/baxmRNA的表達(dá)也有相應(yīng)的變化(P＜0.05)。懸吊組大鼠肺組織iNOS表達(dá)水平明顯高于對照組(P＜0.05),而N-乙酰半胱氨酸干預(yù)組大鼠肺組織iNOS表達(dá)水平明顯低于7天懸吊組(P＜0.05),大鼠肺組織iNOS mRNA表達(dá)也有相應(yīng)的變化(P＜0.05)。同樣,銀杏葉提取物干預(yù)組大鼠肺組織細(xì)胞凋亡指數(shù)(8.1%±1.7%)顯著低于尾懸吊組(P＜0.05),其bcl-2/baxmRNA的表達(dá)也有相應(yīng)的變化(P＜0.05)。而且銀杏葉提取物干預(yù)組大鼠肺組織iNOS表達(dá)水平也明顯低于7天懸吊組(P＜0.05),其iNOS mRNA表達(dá)也有相應(yīng)的變化(P＜0.05)。 結(jié)論: 模擬失重后大鼠肺組織蛋白組學(xué)發(fā)生變化,有些蛋白表達(dá)上調(diào),而有些蛋白表達(dá)下調(diào)甚至消失,影響細(xì)胞能量代謝、應(yīng)激與炎癥反應(yīng)、細(xì)胞損傷與修復(fù)以及細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)等細(xì)胞功能活動,在失重所致肺組織損傷中發(fā)揮著重要的作用。模擬失重時(shí)大鼠肺組織細(xì)胞凋亡水平增高,iNOS表達(dá)水平也增高,而N-乙酰半胱氨酸和銀杏葉提取物均可抑制模擬失重大鼠肺組織的細(xì)胞凋亡過度,并能降低iNOS的表達(dá)水平。
[Abstract]:Objective:
Weightlessness can cause lung function affected, and can cause a certain degree of lung injury. In this experiment, using weight loss method of rat tail suspension model, simulated weightlessness in rats after lung tissue proteome changes, in-depth understanding of the mechanism of lung injury induced by weightlessness; at the same time choose N- acetylcysteine and Ginkgo biloba extract drug intervention, observation of rat lung cell apoptosis and expression of nitric oxide changes, and provide a theoretical basis for the drug loss of lung injury induced by protection.
Method:
Study on the 1. protein group: 20 healthy male S-D rats, weighing 180-220g, were randomly divided into 7 days of simulated weightlessness and 2 normal control group, the tail suspended -30 degrees as the simulated weightlessness model. At the end of the tail suspension, with 10% chloral hydrate (30ml/kg) intraperitoneal injection of anesthesia, thoracic cavity open lung tissue and lung tissue protein extraction and two-dimensional electrophoresis using after protein extraction (2-DE) to establish differential expression patterns, by gel imaging analysis and artificial comparison, determine the different protein spots, and then for each different protein spots were in gel digestion, using HDMS liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis combined with the identification of protein database retrieval. Finally the understanding and detailed classification of biological functions of proteins, further study on their function.
Study on the intervention of 2. drugs: 40 healthy male S-D rats, weighing 180-220g, were randomly divided into 7 days of simulated weightlessness, normal control, intervention N- N-acetylcysteine and Ginkgo biloba extracts 4 groups by tail suspension -30 degrees as the simulated weightlessness model. At the end of the tail suspension, with 10% chloral hydrate (30ml/kg) intraperitoneal injection after anesthesia, open the chest to remove the lung tissue paraffin sections. In situ apoptosis assay, cell apoptosis in pulmonary tissues of rats were observed by immunohistochemistry and in situ hybridization, inducible nitric oxide synthase (iNOS) and Bcl-2, Bax and iNOS change mRNA.
Result:
The changes of 1. groups: the application of 2-DE to protein suspension group and control group rat lung tissue samples were separated and compared, the establishment of differential expression patterns, by gel imaging analysis and manual comparison, determine the different protein spots 17, of which 13 up-regulated and 3 down regulated, 1 disappeared. The isoelectric point distribution in 4.65-8.61, the molecular weight range of 15972-60317, the maximum expression of 6.9 times, the lowest down to 0.1 times. After in gel digestion and mass spectrometry, we identified 17 proteins, including 13 up-regulated and 3 down regulated, 1 disappeared, 5 related to cell metabolism, 4 associated with cell function 3, related to cell structure proteins, 2 related protein degradation system, 3 related to signal transduction. They may be cell energy metabolism, stress and inflammatory reaction, cell damage and repair, intracellular signal transduction and other cell function related activities in cells The immune response, cell apoptosis and other aspects play an important role.
2. drug intervention results: the apoptosis index of lung tissue suspension group rats (23.1% + 2.6%) was significantly higher than the normal control group (2.2% + 1.1%) (P < 0.05), while the N- N-acetylcysteine intervention index group rats lung tissue apoptosis (7.5% + 1.6%) was significantly lower than that of the tail suspension group (P < 0.05). The expression of bcl-2/baxmRNA in lung tissue of rats with corresponding changes (P < 0.05). The level was significantly higher than the control group iNOS expression in lung tissue of rats suspension (P < 0.05), while the N- acetylcysteine intervention in lung tissue of iNOS rats was significantly lower than the expression level of the 7 day suspension group (P < 0.05), rats the lung tissue iNOS mRNA expression also has corresponding change (P < 0.05). Similarly, Ginkgo biloba extract intervention index of rats lung tissue apoptosis (8.1% + 1.7%) was significantly lower than that of the tail suspension group (P < 0.05), the expression of bcl-2/baxmRNA also have corresponding change (P < 0.05) and Ginkgo biloba extract intervention group The expression level of iNOS in rat lung tissue was also significantly lower than that in the 7 day suspension group (P < 0.05), and the expression of iNOS mRNA also had a corresponding change (P < 0.05).
Conclusion:
After simulated weightlessness rats lung tissue proteome changes, expression of some proteins, and some protein expression decreased or disappeared, cell energy metabolism, stress and inflammatory reaction, cell damage and repair and intracellular signal transduction cell function activity, plays an important role in the weight loss caused by lung injury. Simulated weightlessness rat lung tissue cell apoptosis increased, the expression level of iNOS increased, while N- acetylcysteine and Ginkgo biloba extract can inhibit the apoptosis of simulated weightlessness rat lung tissue over expression level and lower iNOS.

【學(xué)位授予單位】：中國人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R852.22

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