本文選題：周期性機(jī)械牽張 + 肺損傷�。� 參考：《山東大學(xué)》2013年碩士論文

【摘要】：背景與目的目前臨床上救治危重患者的重要措施之一是機(jī)械通氣(mechanical ventilation, MV),及早對患者進(jìn)行機(jī)械通氣不但可以糾正難治性低氧血癥,而且對防治肺泡塌陷以及對抗肺水腫也具有重要作用。但是機(jī)械通氣也伴隨著許多并發(fā)癥,如機(jī)械通氣相關(guān)性肺損傷(ventilation-induced lung injury, VILI),目前越來越多的研究人員對此并發(fā)癥高度關(guān)注。參與VILI的細(xì)胞種類有不少,研究較多的有中性粒細(xì)胞、毛細(xì)血管內(nèi)皮細(xì)胞、肺泡上皮細(xì)胞等,而目前的研究熱點(diǎn)是VILI中肺泡巨噬細(xì)胞(Alveolar macrophages, AM)所產(chǎn)生的作用。有研究發(fā)現(xiàn),在生理情況下AM分泌的細(xì)胞因子的種類和數(shù)量都十分有限,但在潮氣量通過機(jī)械牽張導(dǎo)致肺損傷(即傷害性機(jī)械通氣)的病理情況下,AM活化后可分泌大量的炎性細(xì)胞因子、趨化因子、炎性介質(zhì)等多種生物活性物質(zhì)而導(dǎo)致肺損傷。本文中研究的TLR4便是主要存在于哺乳動物肺臟的巨噬細(xì)胞表面,它是Toll樣受體家族的成員之一,是重要的固有免疫分子。由于在體實(shí)驗(yàn)受神經(jīng)體液等多種因素的調(diào)節(jié),單純的機(jī)械牽張對大鼠肺泡巨噬細(xì)胞上TLR4受體的影響尚不是很清楚。我們的實(shí)驗(yàn)?zāi)康脑谟谔接憴C(jī)械牽張對大鼠AM Toll樣受體4(TLR4)表達(dá)的影響。 方法用預(yù)冷的無菌的PBS-H(10%小牛血清的PBS和含10U/ml肝素)對大鼠進(jìn)行支氣管肺泡充分灌洗,然后將肺泡灌洗液中的AM進(jìn)行分離和純化,最后收集的貼壁細(xì)胞便是AM。本實(shí)驗(yàn)將培養(yǎng)后的AM隨機(jī)分為3組(n=6)：機(jī)械牽張6h組、機(jī)械牽張8h組和靜止對照組。機(jī)械牽張組細(xì)胞采用美國Flexercell公司生產(chǎn)的Flexercell4000TTM應(yīng)力加載系統(tǒng),施加的牽張力大小為能夠使變形膜拉伸30%,牽張頻率為30次/min,牽拉：松弛=1：1,牽張時間分別為6h和8h。靜止對照組細(xì)胞除了未施加周期性牽張外,其余條件均同牽張8h組。逆轉(zhuǎn)錄酶PCR (reverse transcriptase PCR, RT-PCR)檢測AM上的TLR4mRNA的表達(dá)；ELISA檢測巨噬細(xì)胞炎性蛋白2(macrophage inflammatory protein2, MIP-2)的濃度；免疫細(xì)胞化學(xué)法檢測AM上TLR4蛋白的表達(dá)。最后的數(shù)據(jù)用統(tǒng)計學(xué)進(jìn)行處理。 結(jié)果AM的TLR4RT-PCR結(jié)果用TLR4mRNA/ACTINmRNA的OD比值表示,與對照組比,機(jī)械牽張組TLR4mRNA條的表達(dá)差異有統(tǒng)計學(xué)意義(P0.01),機(jī)械牽張6h組與8h組表達(dá)差異無顯著性；機(jī)械牽張組細(xì)胞培養(yǎng)液中MIP-2的濃度升高差異有顯著性(P0.01),機(jī)械牽張6h組與8h組濃度差異無顯著性；AM的TLR4免疫細(xì)胞化學(xué)結(jié)果以實(shí)驗(yàn)組TLR4的吸光度值-陰性對照組TLR4的吸光度值(△TLR4)表示,與對照組比,牽張組/XTLR4的升高差異有顯著性(P0.01),機(jī)械牽張6h組與8h組的表達(dá)無統(tǒng)計學(xué)差異。 結(jié)論機(jī)械牽張導(dǎo)致了大鼠肺泡巨噬細(xì)胞TLR4和MIP-2表達(dá)上調(diào)。
[Abstract]:Background & objective at present, one of the most important measures to treat critical patients is mechanical ventilation (MVV). Early mechanical ventilation can not only correct refractory hypoxemia.It also plays an important role in the prevention and treatment of alveolar collapse and the prevention of pulmonary edema.However, mechanical ventilation is accompanied by many complications, such as ventilation-induced lung injury.At present, more and more researchers are paying close attention to this complication.There are many kinds of cells involved in VILI, such as neutrophils, capillary endothelial cells, alveolar epithelial cells and so on. The current research focus is on the role of alveolar macrophages (AMs) in VILI.Some studies have found that the number and variety of cytokines secreted by AM are very limited under physiological conditions.However, under the pathological condition that the tidal volume leads to lung injury (i.e. noxious mechanical ventilation) by mechanical stretch, AM can secrete a large number of inflammatory cytokines, chemokines, inflammatory mediators and other biological active substances, which can lead to lung injury.TLR4, which is mainly found on the surface of macrophages in mammalian lungs, is a member of the Toll like receptor family and an important innate immune molecule.The effect of mechanical distraction on the TLR4 receptor on alveolar macrophages in rats is not clear due to in vivo regulation by neurohumoral and other factors.Our aim was to investigate the effect of mechanical stretch on the expression of AM Toll like receptor 4 TLR4 in rats.Methods the bronchoalveolar lavage of rats was carried out with PBS and 10U/ml heparin in 10% calf serum of precooled aseptic PBS-Hn. The AM in alveolar lavage fluid was isolated and purified. The last adherent cell was Am.The cultured AM was randomly divided into 3 groups: 6 h mechanical distraction group, 8 h mechanical distraction group and a static control group.The cells in the mechanical stretch group were subjected to Flexercell4000TTM stress loading system produced by Flexercell Company in the United States. The tensile force was applied to make the deformable membrane stretch 30 times per minute. The stretch frequency was 30 times / min. The stretch time was 6 h and 8 h respectively. The stretching time was 1: 1. The stretch time was 6 h and 8 h, respectively.The cells in the static control group were all the same as those in the 8 h group except that the cells were not subjected to cyclic distraction.Reverse transcriptase PCR (RT-PCR) was used to detect the expression of TLR4mRNA in AM. The concentration of 2(macrophage inflammatory protein 2 (MIP-2) was detected by Elisa, and the expression of TLR4 on AM was detected by immunocytochemistry.The final data is processed statistically.Results the TLR4RT-PCR results of AM were expressed by the OD ratio of TLR4mRNA/ACTINmRNA. Compared with the control group, the expression of TLR4mRNA in the mechanical distraction group was significantly different from that in the control group (P 0.01), but there was no significant difference between the mechanical distraction group and the 8 h group.There was significant difference in the concentration of MIP-2 in the cell culture medium of mechanical distraction group (P 0.01). There was no significant difference in the concentration of TLR4 between 6 h group and 8 h group of mechanical distraction group. The results of TLR4 immunocytochemistry of the experimental group were as follows: the absorbance of TLR4 in the experimental group and the value of TLR4 in the negative control group.The absorbance value (TLR4) indicates,Compared with the control group, the increase of XTLR4 in the distraction group was significantly higher than that in the control group (P 0.01), but there was no significant difference in the expression of XTLR4 between the 6 h group and the 8 h group.Conclusion Mechanical distraction leads to upregulation of TLR4 and MIP-2 expression in alveolar macrophages.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R563

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張燕;武慶平;姚尚龍;;周期性機(jī)械牽張對肺泡Ⅱ型上皮細(xì)胞Cyr61表達(dá)的影響[J];中國組織化學(xué)與細(xì)胞化學(xué)雜志;2011年01期

2 盛金良;陳創(chuàng)夫;楊霞;王遠(yuǎn)志;張輝;;綿羊Toll樣受體家族在肺泡巨噬細(xì)胞的分布及脂多糖(LPS)刺激對TLR2、TLR4表達(dá)的影響[J];華北農(nóng)學(xué)報;2010年01期

3 張宏,孟亞娟,斯琴,王士雯,孫仁宇;銀杏葉提取物對脂多糖誘導(dǎo)大鼠急性肺損傷的保護(hù)作用[J];基礎(chǔ)醫(yī)學(xué)與臨床;2002年02期

4 任大賓,杜燁瑋,張健,孫仁宇,王士雯;靜脈注射脂多糖上調(diào)小鼠肺及肝CD14和Toll-like受體4表達(dá)[J];基礎(chǔ)醫(yī)學(xué)與臨床;2005年04期

5 王月蘭,姚尚龍;磷脂酰肌醇-3-激酶參與過度機(jī)械通氣誘導(dǎo)的核因子-κB激活[J];臨床麻醉學(xué)雜志;2005年09期

6 宋秀梅;王月蘭;劉洋;;不同潮氣量機(jī)械通氣誘發(fā)的肺損傷大鼠肺組織中CXCR2表達(dá)[J];臨床麻醉學(xué)雜志;2012年09期

7 李錦師;白建文;;Toll樣受體與急性肺損傷研究進(jìn)展[J];上海交通大學(xué)學(xué)報(醫(yī)學(xué)版);2007年07期

8 祝筱梅;劉秀華;蔡莉蓉;徐菲菲;;p38絲裂素活化蛋白激酶介導(dǎo)低氧預(yù)處理誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)的心肌細(xì)胞保護(hù)[J];生理學(xué)報;2006年05期

9 丁寧;肖慧;高巨;許立新;佘守章;;P38及上游激酶MKK6對周期性牽張應(yīng)變誘導(dǎo)的肺泡巨噬細(xì)胞高遷移率族蛋白B1表達(dá)的調(diào)控[J];生理學(xué)報;2009年01期

10 張潁;趙慧娟;;肺泡巨噬細(xì)胞與急性肺損傷[J];山西職工醫(yī)學(xué)院學(xué)報;2009年01期

相關(guān)博士學(xué)位論文 前1條

1 肖曉燕;TLR4在1型糖尿病腎病及TLR3在自身免疫性肝炎中作用的研究[D];山東大學(xué);2010年

相關(guān)碩士學(xué)位論文 前1條

1 李鐵英;糖尿病大鼠肺泡巨噬細(xì)胞TLR4的表達(dá)及對LPS反應(yīng)性的研究[D];中國醫(yī)科大學(xué);2009年

，

本文編號：1742599