周期性机械牵张对大鼠肺泡巨噬细胞TLR4表达的影响

发布时间：2018-04-13 02:39

本文选题：周期性机械牵张 + 肺损伤　；参考：《山东大学》2013年硕士论文

【摘要】：背景与目的目前临床上救治危重患者的重要措施之一是机械通气(mechanical ventilation, MV),及早对患者进行机械通气不但可以纠正难治性低氧血症,而且对防治肺泡塌陷以及对抗肺水肿也具有重要作用。但是机械通气也伴随着许多并发症,如机械通气相关性肺损伤(ventilation-induced lung injury, VILI),目前越来越多的研究人员对此并发症高度关注。参与VILI的细胞种类有不少,研究较多的有中性粒细胞、毛细血管内皮细胞、肺泡上皮细胞等,而目前的研究热点是VILI中肺泡巨噬细胞(Alveolar macrophages, AM)所产生的作用。有研究发现,在生理情况下AM分泌的细胞因子的种类和数量都十分有限,但在潮气量通过机械牵张导致肺损伤(即伤害性机械通气)的病理情况下,AM活化后可分泌大量的炎性细胞因子、趋化因子、炎性介质等多种生物活性物质而导致肺损伤。本文中研究的TLR4便是主要存在于哺乳动物肺脏的巨噬细胞表面,它是Toll样受体家族的成员之一,是重要的固有免疫分子。由于在体实验受神经体液等多种因素的调节,单纯的机械牵张对大鼠肺泡巨噬细胞上TLR4受体的影响尚不是很清楚。我们的实验目的在于探讨机械牵张对大鼠AM Toll样受体4(TLR4)表达的影响。方法用预冷的无菌的PBS-H(10%小牛血清的PBS和含10U/ml肝素)对大鼠进行支气管肺泡充分灌洗,然后将肺泡灌洗液中的AM进行分离和纯化,最后收集的贴壁细胞便是AM。本实验将培养后的AM随机分为3组(n=6)：机械牵张6h组、机械牵张8h组和静止对照组。机械牵张组细胞采用美国Flexercell公司生产的Flexercell4000TTM应力加载系统,施加的牵张力大小为能够使变形膜拉伸30%,牵张频率为30次/min,牵拉：松弛=1：1,牵张时间分别为6h和8h。静止对照组细胞除了未施加周期性牵张外,其余条件均同牵张8h组。逆转录酶PCR (reverse transcriptase PCR, RT-PCR)检测AM上的TLR4mRNA的表达；ELISA检测巨噬细胞炎性蛋白2(macrophage inflammatory protein2, MIP-2)的浓度；免疫细胞化学法检测AM上TLR4蛋白的表达。最后的数据用统计学进行处理。结果AM的TLR4RT-PCR结果用TLR4mRNA/ACTINmRNA的OD比值表示,与对照组比,机械牵张组TLR4mRNA条的表达差异有统计学意义(P0.01),机械牵张6h组与8h组表达差异无显著性；机械牵张组细胞培养液中MIP-2的浓度升高差异有显著性(P0.01),机械牵张6h组与8h组浓度差异无显著性；AM的TLR4免疫细胞化学结果以实验组TLR4的吸光度值-阴性对照组TLR4的吸光度值(△TLR4)表示,与对照组比,牵张组/XTLR4的升高差异有显著性(P0.01),机械牵张6h组与8h组的表达无统计学差异。结论机械牵张导致了大鼠肺泡巨噬细胞TLR4和MIP-2表达上调。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563

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