發(fā)布時(shí)間：2018-03-24 13:41

  本文選題：急性肺損傷　切入點(diǎn)：脂多糖　出處：《南華大學(xué)》2016年碩士論文

【摘要】：急性肺損傷(ALI)發(fā)病率高,其發(fā)病機(jī)制復(fù)雜。當(dāng)急性肺損傷進(jìn)一步加重則會發(fā)展為急性呼吸窘迫綜合征(ARDS),其致死率高達(dá)40%。研究發(fā)現(xiàn)促炎細(xì)胞因子與抗炎細(xì)胞因子之間的失衡可能是ALI的主要發(fā)病機(jī)制,革蘭陰性細(xì)菌感染和內(nèi)毒素血癥是ALI/ARDS的常見致病因素。膽堿能抗炎通路是近年來的研究熱點(diǎn),該通路由迷走神經(jīng)及神經(jīng)遞質(zhì)乙酰膽堿和膽堿能受體構(gòu)成,具有調(diào)節(jié)全身性炎癥反應(yīng)的作用,主要通過迷走神經(jīng)抑制炎癥因子的合成和釋放,從而抑制機(jī)體炎癥反應(yīng)。其中α7煙堿型乙酰膽堿受體(α7nAChR)在膽堿能抗炎通路中起重要的作用。大量動物實(shí)驗(yàn)結(jié)果表明使用α7nAChR激動劑能在創(chuàng)傷、敗血癥及類風(fēng)濕關(guān)節(jié)炎等全身性炎癥中抑制炎癥因子的生成和釋放。GTS-21是α7nAChR特異性激動劑,在脂多糖誘導(dǎo)的急性肺損傷是否發(fā)揮著抗炎作用及其作用機(jī)制,目前國內(nèi)報(bào)道較少。目的觀察α7nAChR與炎癥因子IL-1β、IL-18、HMGB1在脂多糖誘導(dǎo)小鼠急性肺損傷模型中的動態(tài)表達(dá),研究α7nAChR激動劑GTS-21對脂多糖誘導(dǎo)小鼠急性肺損傷模型的保護(hù)作用,探索GTS-21在急性肺損傷中的抗炎作用機(jī)制,為治療急性肺損傷提供新策略。方法實(shí)驗(yàn)分為兩個部分。第一部分:α7nAChR在急性肺損傷中的作用機(jī)制研究。將50只C57BL/6小鼠隨機(jī)分成5組:正常對照組、急性肺損傷3小時(shí)組、急性肺損傷6小時(shí)組、急性肺損傷12小時(shí)組、急性肺損傷24小時(shí)組,每組10只。正常對照組腹腔注射等量生理鹽水,1小時(shí)后斷頸法處死小鼠;余組采用30mg/kg的LPS腹腔注射造模并在LPS注射相應(yīng)時(shí)間后處死存活小鼠。每組選取8個標(biāo)本留取肺組織檢測小鼠左肺的濕/干(W/D)比重;HE染色,光鏡下觀察肺臟病理變化;運(yùn)用ELISA分別檢測右肺組織勻漿中的IL-1β、IL-18、HMGB1水平表達(dá);采用Real-Time PCR檢測肺組織α7nAChR m RNA的表達(dá)。第二部分,GTS-21靶向干預(yù)觀察。將63只小鼠隨機(jī)分成4組:正常對照組(NC組,n=9),急性肺損傷模型組(LPS組,n=9),藥物對照組(GTS-21組,n=9),GTS-21干預(yù)組(LPS+GTS-21組,n=36)。NC組給予腹腔注射磷酸鹽緩沖液(PBS);LPS組予以腹腔注射30mg/kg的LPS造模;GTS-21組在PBS注射前30分鐘腹腔注射3 mg/kg的GTS-21;LPS+GTS-21組在LPS注射前30分鐘腹腔注射3 mg/kg的GTS-21。正常對照組、急性肺損傷模型組、藥物對照組均在PBS注射或LPS注射后6小時(shí)斷頸處死小鼠。GTS-21干預(yù)組分別在LPS腹腔注射后3小時(shí)、6小時(shí)、12小時(shí)、24小時(shí)處死小鼠。每組選取8個標(biāo)本送檢,觀察各組肺濕/干(W/D)比重、肺組織病理變化、肺組織勻漿中的IL-1β、IL-18、HMGB1水平表達(dá)及肺組織α7nAChR m RNA的表達(dá)。結(jié)果第一部分(1)與正常對照組比較,急性肺損傷組肺臟W/D比重明顯增高(3h:4.49±0.16 vs 4.06±0.11,6h:5.57±0.18 vs 4.06±0.11,12h:5.32±0.14 vs 4.06±0.11,24h:5.25±0.20 vs 4.06±0.11,均p0.01);肺臟病理變化明顯加重;IL-1β水平明顯升高(3h:2875.28±169.95 vs555.73±132.89,6h:3136.51±130.56 vs 555.73±132.89,12h:2475.36±190.50 vs 555.73±132.89,24h:256.83±413.40 vs 555.73±132.89,均p0.01);IL-18水平明顯升高(3h:176.72±20.86 vs 144.92±21.43,6h:200.96±21.16 vs 144.92±21.43,12h:183.82±7.76 vs 144.92±21.43,24h:159.59±16.09 vs 144.92±21.43,p0.05);HMGB1水平亦明顯升高(3h:3.99±0.82 vs 1.88±0.73,6h:5.9±1.33 vs 1.88±0.73,12h:7.85±0.65 vs 1.88±0.73,24h:6.49±0.51 vs 1.88±0.73,p0.01);肺臟W/D比重、肺臟病理評分、肺組織勻漿中IL-1β、IL-18的水平在6小時(shí)達(dá)高峰,HMGB1在12小時(shí)達(dá)高峰,組間差異具有統(tǒng)計(jì)學(xué)意義(p0.05)。(2)與正常對照組比較,急性肺損傷組肺組織勻漿中α7nAChR m RNA表達(dá)呈逐漸降低趨勢,差異均有統(tǒng)計(jì)學(xué)意義。(3h:1.57±0.16 vs 2.06±0.29,6h:0.79±0.13 vs 2.06±0.29,12h:0.65±0.12 vs 2.06±0.29,24h:0.19±0.04 vs 2.06±0.29,p0.01)。第二部分(1)與正常對照組比較,藥物對照組小鼠的肺W/D比重及肺臟病理評分及IL-1β、IL-18、HMGB1水平及α7nAChR m RNA的表達(dá)的差異均無明顯統(tǒng)計(jì)學(xué)意義(p0.05)。(2)與急性肺損傷組比較,GTS-21干預(yù)組的肺組織的α7nAChR m RNA表達(dá)明顯增高(6h:1.23±0.41 vs 0.81±0.13,p0.05)。(3)與急性肺損傷組相對應(yīng)時(shí)間點(diǎn)比較,GTS-21干預(yù)組的肺W/D比重明顯改善(3h:4.20±0.13 vs 4.49±0.16,6h:5.21±0.11 vs 5.57±0.18,12h:5.17±0.13 vs5.32±1.14,24h:5.06±0.13 vs 5.25±0.20,p0.05);肺組織病理評分明顯降低(3h:5.25±0.46 vs 6.00±0.75,6h:10.5±0.92 vs 12.37±1.18,12h:7.25±0.71 vs 8.50±1.19,24h:5.75±1.03 vs 7.25±1.58,p0.05);IL-1β水平明顯降低(3h:2585.10±143.27 vs 2875.28±169.95,6h:1899.01±121.74 vs 3136.51±130.56,12h:1640.89±211.90 vs 2475.36±190.50,24h:1624.06±130.13 vs 2256.83±413.40,p0.01);IL-18水平明顯降低(3h:155.73±13.90 vs 176.72±20.86,6h:164.05±15.20vs 200.96±21.16,12h:151.83±22.06 vs 183.82±7.76,24h:132.45±13.05 vs 159.59±16.09,p0.05);HMGB1水平明顯降低(3h:3.16±0.61 vs 3.99±0.82,6h:4.07±1.40 vs 5.9±1.33,12h:5.37±1.03 vs7.85±0.65,24h:5.12±1.31 vs 6.49±0.51,p0.05)。結(jié)論(1)α7nAChR在脂多糖誘導(dǎo)的急性肺損傷中發(fā)揮重要作用。(2)GTS-21對脂多糖誘導(dǎo)的急性肺損傷起保護(hù)作用。
[Abstract]:Acute lung injury (ALI) has high incidence, its pathogenesis is complex. When the acute lung injury will further aggravate the development of acute respiratory distress syndrome (ARDS), the mortality rate as high as 40%. study found that the unbalance between pro-inflammatory cytokines and anti-inflammatory cytokines may be the main pathogenesis of ALI, gram negative bacterial infection and endotoxemia is a common pathogenic factor of ALI/ARDS. The cholinergic anti-inflammatory pathway is a hot research topic in recent years, the path of the vagus nerve and the neurotransmitter acetylcholine and cholinergic receptor, can regulate the systemic inflammatory response, inhibit the synthesis and release of inflammatory factors, mainly through the vagus nerve, thereby inhibiting the inflammatory reaction. The alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR) plays an important role in the cholinergic anti-inflammatory pathway. A large number of animal experiments show that the use of alpha 7nAChR agonists in a Injury, inflammation and sepsis, rheumatoid arthritis and systemic in inhibition of cytokine production and release of.GTS-21 alpha 7nAChR specific agonists in lipopolysaccharide induced acute lung injury or play an anti-inflammatory effect and mechanism, currently reported. Objective To observe the effect of alpha 7nAChR and inflammatory cytokine of IL-1, IL-18, HMGB1 in lipopolysaccharide the dynamic model of acute lung injury in mice induced by the expression of protective effect of alpha 7nAChR agonist GTS-21 on lipopolysaccharide induced acute lung injury model of mice, to explore the anti-inflammatory mechanism of GTS-21 in acute lung injury, for the treatment of acute lung injury and provide new strategies. Methods the experiment was divided into two parts. The first part: the mechanism research alpha 7nAChR in acute lung injury. 50 C57BL/6 mice were randomly divided into 5 groups: normal control group, 3 hours group, 6 hours group acute lung injury, acute lung injury, acute lung Injury 12 hours group, 24 hours group acute lung injury, 10 rats in each group. The normal control group were injected with saline, 1 hours after the mice were killed by cervical dislocation; more than 30mg/kg group by intraperitoneal injection of LPS and LPS in rats were sacrificed after injection of the corresponding time survival in mice. Each group selected 8 samples of lung specimens detection of mice left lung wet / dry weight (W/D); HE staining, to observe the pathologic changes under light microscope were detected by ELISA; the right lung tissue homogenates of IL-1 beta, IL-18, HMGB1 expression by PCR; expression of Real-Time in lung tissue was measured by alpha 7nAChR RNA m. In the second part, GTS-21 targeted intervention observation. 63 mice were randomly divided into 4 groups: normal control group (group NC, n=9), acute lung injury model group (group LPS, n=9), the drug control group (group GTS-21, n=9), GTS-21 group (group LPS+GTS-21, n=36).NC group were given intraperitoneal injection of phosphate buffer (PBS) LPS group was given intraperitoneal; Injection of 30mg/kg LPS model; GTS-21 group was injected PBS 30 minutes before the intraperitoneal injection of 3 mg/kg GTS-21; LPS+GTS-21 group was injected LPS 30 minutes before the intraperitoneal injection of 3 mg/kg GTS-21. normal control group, model group, acute lung injury, drug control group were injected into PBS or LPS were sacrificed 6 hours after injection of.GTS-21 in mice the intervention group were intraperitoneal injection of LPS after 3 hours, 6 hours, 12 hours, 24 hours. The mice were killed in each group were randomly selected 8 specimens were observed, lung wet / dry weight (W/D), the pathological changes of lung tissue, lung tissue homogenate of IL-1 beta, IL-18, HMGB1 expression level in pulmonary tissue and expression of alpha the 7nAChR m RNA. The results of the first part (1) compared with the normal control group, acute lung injury group W/D a significantly higher proportion of lung (3h:4.49 + 0.16 vs 4.06 + 0.11,6h:5.57 0.18 + vs 4.06 + 0.11,12h:5.32 0.14 + vs 4.06 + 0.11,24h:5.25 + 0.20 vs 4.06 + 0.11, P0.01); lung disease Physical changes significantly increased; IL-1 beta levels were significantly increased (3h:2875.28 169.95 + vs555.73 + 132.89,6h:3136.51 + 130.56 vs 555.73 + 132.89,12h:2475.36 190.50 + vs 555.73 + 132.89,24h:256.83 + 413.40 vs 555.73 + 132.89, P0.01); the IL-18 level was significantly higher in 3h:176.72 (+ 20.86 vs 144.92 + 21.43,6h:200.96 21.16 + vs 144.92 + 21.43,12h:183.82 7.76 + vs 144.92 + 21.43,24h:159.59 144.92 + 16.09 vs + 21.43, P0.05); the HMGB1 level also increased significantly (3h:3.99 0.82 + vs 1.88 + 0.73,6h:5.9 + 1.33 vs + 1.88 0.73,12h:7.85 + 0.65 vs + 1.88 0.73,24h:6.49 + 0.51 vs 1.88 + 0.73, P0.01); W/D lung weight, lung pathological score, lung tissue homogenates of IL-1 beta, IL-18 level reached the peak in 6 hours, HMGB1 reached the peak in 12 hours, the difference was statistically significant (P0.05). (2) compared with normal control group, lung tissue homogenates of alpha 7n in acute lung injury The expression of AChR m RNA was gradually decreased, the differences were statistically significant. (3h:1.57 + 0.16 vs 2.06 + 0.29,6h:0.79 0.13 + vs 2.06 + 0.29,12h:0.65 0.12 + vs 2.06 + 0.29,24h:0.19 + 0.04 vs 2.06 + 0.29, P0.01). The second part (1) compared with the normal control group, drug control group and the proportion of W/D in the lungs of mice lung pathological score and IL-1 beta, IL-18, HMGB1 and 7nAChR m level alpha RNA expression difference was statistically significant (P0.05). (2) compared with the acute lung injury group, GTS-21 intervention group lung tissue alpha 7nAChR m expression of RNA was significantly higher (6h: 1.23 + 0.41 vs 0.81 + 0.13, P0.05). (3) and acute lung injury group of corresponding time points, GTS-21 in the intervention group significantly improved the proportion of lung W/D (3h:4.20 + 0.13 vs 4.49 + 0.16,6h:5.21 + 0.11 vs + 5.57 0.18,12h:5.17 + 0.13 vs5.32 + 5.25 1.14,24h:5.06 + 0.13 vs + 0.20, P0.05); lung tissue pathological score of Ming Dynasty Significant decrease (3h:5.25 + 0.46 vs 6 + 0.75,6h:10.5 0.92 + vs 12.37 + 1.18,12h:7.25 0.71 + vs 8.50 + 1.19,24h:5.75 + 1.03 vs 7.25 + 1.58, P0.05); beta IL-1 levels were significantly lower (3h:2585.10 143.27 + vs 2875.28 + 169.95,6h:1899.01 121.74 + vs 3136.51 + 130.56,12h:1640.89 211.90 + vs 2475.36 + 190.50,24h:1624.06 + 130.13 vs 2256.83 + 413.40. P0.01); IL-18 significantly decreased (3h:155.73 + 13.90 vs 176.72 + 20.86,6h:164.05 200.96 + 15.20vs + 21.16,12h:151.83 + 22.06 vs 183.82 + 7.76,24h:132.45 + 13.05 vs 159.59 + 16.09, P0.05); HMGB1 significantly decreased (3h:3.16 + 0.61 vs 3.99 + 0.82,6h:4.07 1.40 + vs 5.9 + 1.33,12h:5.37 1.03 + vs7.85 + 0.65,24h:5.12 + 1.31 vs + 6.49 0.51, P0.05). Conclusion (1) alpha 7nAChR play an important role in acute lung injury induced by lipopolysaccharide. (2) GTS-21 on lipopolysaccharide induced acute lung injury It plays a protective role.

【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R563.8

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳英華;呂嘉文;翟啟良;謝哲凡;胡國棟;;LPS直接及間接作用致小鼠急性肺損傷模型的建立[J];中國臨床解剖學(xué)雜志;2015年04期

2 陳濤;丁嵐;;膿毒癥炎癥調(diào)控的研究進(jìn)展[J];實(shí)用臨床醫(yī)學(xué);2015年06期

3 趙利利;戎浩;孫芳云;;內(nèi)毒素致急性肺損傷的發(fā)病機(jī)制研究進(jìn)展[J];實(shí)用藥物與臨床;2015年04期

4 魏婷婷;李雙杰;;α7煙堿型乙酰膽堿受體的抗炎機(jī)制研究進(jìn)展[J];國際兒科學(xué)雜志;2014年04期

5 金發(fā)光;;急性肺損傷的診治研究現(xiàn)狀及進(jìn)展[J];中華肺部疾病雜志(電子版);2013年01期

6 張祺嘉鈺;孫毅;胡銳;張恩戶;王敏;楊洋;;內(nèi)毒素不同給藥途徑致急性肺損傷模型的研究[J];現(xiàn)代中醫(yī)藥;2013年01期

7 鄭暉;許紹發(fā);賈鴻彥;古淑香;;膽堿能抗炎通路對內(nèi)毒素復(fù)合油酸致大鼠急性肺損傷的影響[J];首都醫(yī)科大學(xué)學(xué)報(bào);2012年03期

8 張霄旦;趙擎宇;;膽堿能抗炎通路的機(jī)制及其在膿毒癥的應(yīng)用[J];國際病理科學(xué)與臨床雜志;2011年06期

9 李超然;王智剛;朱運(yùn)奎;;內(nèi)毒素所致急性肺損傷發(fā)病機(jī)制的研究進(jìn)展[J];中國醫(yī)藥科學(xué);2011年10期

10 王金榮,王宏偉;炎性反應(yīng)反射—膽堿能抗炎通路[J];中華神經(jīng)醫(yī)學(xué)雜志;2004年04期

，

本文編號：1658548