内毒素诱导急性肺损伤中热休克蛋白的表达及大黄对HSP70表达的调控

发布时间：2018-01-14 11:10

本文关键词：内毒素诱导急性肺损伤中热休克蛋白的表达及大黄对HSP70表达的调控　出处：《中国医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 内毒素诱导急性肺损伤中热休克蛋白的表达及大黄对HSP70表达的调控前言急性呼吸窘迫综合症(ARDS)是临床常见的危急重症,其病理特征是中性粒细胞在肺内聚集、血管内凝血、肺毛细血管通透性增加致肺水肿,从而导致低氧血症甚至死亡。热休克蛋白(heat shock protein.HSP)是一种应激蛋白(sress protein.SP),同时它也是一种内源性的保护物质,其中HSP70为最保守、最主要的一类,在大多数生物中含量最多,在细胞应激后生成最为显著。另外,研究表明大黄对LPS诱导的ALI具有保护作用,可以抑制一氧化氮生成和iNOS活性,并且可以降低PLA2(磷脂酶A2)和血小板活化因子(PAF)的活性,从而起到保护肺脏,减轻肺损伤LPS诱导的ALI的作用。大黄对肺脏的保护作用是否与HSP家族成员有关,即大黄是否通过诱导肺内HSP70的表达水平来保护肺组织,目前国内外尚未见报道,而且HSP70表达水平在LPS诱导的ALI的动物模型中的时间规律性尚且不清。本实验应用免疫组织化学染色技术和蛋白印迹试验等技术进行研究大黄,并对大黄预防组和大黄治疗组LPS诱导的肺损伤组织中HSP70的表达部位及表达水平进行对比,从而证实大黄对LPS诱导的ALI的保护机制可能与HSP70有关,并为临床上应用大黄治疗急性肺损伤提供理论依据。材料和方法动物模型的建立及分组：健康wistar大鼠60只,随机分成对照组、ALI组、大黄预防组、大黄治疗组4组,每组15只。麻醉后,颈内静脉给0.9％生理盐水或给,LPS,于不同时间,腹主动脉取血做血气分析,后脱颈椎处死动物,并取双侧肺脏标本,进行肺系数测定,而后进行HE染色及免疫组织化学染色(SP法)以及Western-Blotting测定蛋白表达水平。结果判定：免疫组织化学阳性物质表达在胞浆或胞核中,阳性判定应用阳性细胞计数：显微镜×400倍率下每张切片中随机选择10个高倍视野,计算其中阳性细胞数和总细胞数,求出百分比。蛋白印迹结果进行条带灰度测定。阳性细胞百分比、蛋白印迹灰度值、血气分析结果和肺系数等数据资料均以均数±标准差((?)±s)表示,组内不同时间比较均采用t检验。实验结果 1、血气分析(PO2) 对照组各时间段无明显差异,LPS组较对照组下降,以1h、2h为明显。预防用药组对LPS诱导的肺损伤大鼠的PO2改善较明显。 2、肺系数损伤组大鼠肺系数增高,与对照组存在显著性差异,预防用药组和治疗用药组肺系数低于损伤组,存在显著性差异。 3、大体标本和HE染色结果 LPS组肺组织可见斑片状坏死,整个肺充血、肺间质水肿,肺泡间隔增宽,肺泡腔内弥漫性出血及散在支气管内出血,预防用药及治疗用药组肺表面充血有明显减轻,斑片状坏死消失,以预防用药组为明显。LPS组肺泡腔内可见大量红细胞和中性粒细胞浸润,血管及支气管周围组织疏松水肿,有较多炎细胞浸润，以中性粒细胞为主,晚期可见散在透明膜形成。预防用药组和治疗用药组肺脏的组织病理学有明显改善,但仍可见较明显的炎细胞浸润。 4、HSP70免疫组织化学染色结果正常组HSP70主要分布于支气管及细支气管上皮细胞,肺泡细胞和巨噬细胞可见散在阳性着色。LPS组表达增多,可见散在巨噬细胞和浸润的中性粒细胞阳性着色,肺泡上皮细胞阳性数轻度增加,阳性细胞率与对照组相比存在显著性差异。预防用药组与治疗用药组阳性细胞数增加。 5、蛋白质印迹分析(western-bloting方法) HSP70条带位于70KD附近,HSP70蛋白量显示,预防用药组和治疗用药组表达量较对照组和LPS组加深、加宽。并且预防用药组与治疗用药组之间存在显著性差异(P0.01) 讨论急性肺损伤病理特征是中性粒细胞在肺内聚集,血管内凝血,肺毛细血管通透性增加致肺水肿,从而导致低氧血症。LPS还可以增加由其介导的各种生物活性因子的生物效应,加重肺脏损伤和临床症状。热休克蛋白可以提高细胞的应激能力,抵御各种损害因素的影响,起到保护细胞的作用,从而降低动物的死亡率。本实验研究中预防用药组HSP70表达增加,免疫组织化学阳性细胞率和Western结果均高于急性肺损伤组,而且大鼠动脉血氧分压增高、肺系数降低、肺组织损伤的病理学改变较轻,治疗用药组虽有一定程度的改善,但在动脉血氧分压增高、肺系数降低、肺组织损伤的病理学改变和HSP70表达量上较预防用药组差。揭示细胞经大黄预处理后细胞中HSP70合成增加,同时也表明了大黄可能通过诱导HSP70的表达增加来保护损伤的肺组织。本研究的实验结果对临床上应用大黄注射液的治疗的时间上具有指导性意义。结论本实验研究表明,LPS组肺脏损伤较明显,在预防用药组可见明显的肺脏功能的改善,治疗用药组也可见HSP70表达较LPS组增高,并且HSP70在支气管、细支气管粘膜上皮和血管内皮均有强阳性表达,说明了HSP70很可能参与了降低血管通透性和保证气管的正常通气。本实验的研究结果表明在注射LPS之前预先给予大黄注射液可以增加肺组织中HSP70的表达量,具有明显的肺脏保护功能,但在治疗用药组中,可见HSP70表达量的轻度增加,与预防用药组存在显著性差异,说明在诱导ALI同时应用大黄注射液来诱导HSP70表达量的增多并不能明显改善肺脏功能。本研究的实验结果对临床上应用大黄注射液的治疗的时间上具有指导性意义,也说明了大黄对ALI的保护作用很可能是通过诱导HSP70的表达增多而实现的。
[Abstract]:Expression of heat shock protein in acute lung injury induced by endotoxin and the regulation of rhubarb on the expression of HSP70
Preface
Acute respiratory distress syndrome (ARDS) is a common severe disease in clinical practice. Its pathological feature is neutrophil aggregation in the lung, intravascular coagulation and increased pulmonary capillary permeability causing pulmonary edema, leading to hypoxemia and even death.
Heat shock protein (heat shock protein.HSP) is a stress protein (sress protein.SP), protective substances, and it is also a kind of endogenous HSP70, which is the most conservative, one of the largest, most abundant in most organisms, the cell stress generated after the most significant. In addition, studies show that rhubarb has protective effect on LPS induced by ALI can inhibit the production of nitric oxide and iNOS activity, and can reduce the PLA2 (phospholipase A2) and platelet activating factor (PAF) activity, so as to protect the lungs, alleviate the lung injury induced by LPS ALI.
Whether the protective effect of rhubarb on the lung associated with HSP family members, namely, whether through the expression of rhubarb induced pulmonary HSP70 to protect lung tissue has not been reported at home and abroad, and the HSP70 expression level in LPS time of animal model induced by ALI in the study is not clear. In this experiment, immunohistochemical rhubarb immunohistochemical staining and Western blot test, comparing the expression site and HSP70 prevention group and rhubarb treatment group LPS induced lung injury in rhubarb and expression level, so as to prove that the protection mechanism of rhubarb on LPS induced by ALI may be related to HSP70, and in the treatment of acute lung injury and provide a theoretical basis for clinical application rhubarb.
Materials and methods
The establishment of animal models and grouping: 60 healthy Wistar rats, were randomly divided into control group, ALI group, rhubarb prevention group, rhubarb treatment group 4 groups, 15 rats in each group. After anesthesia, the internal jugular vein for 0.9% ns to LPS, or, in different time, aortic blood gas analysis, after removal the cervical executed animal, and bilateral lung specimens, lung coefficient, HE staining and immunohistochemical staining (SP method) and then determination of Western-Blotting protein expression.
Results: immunohistochemical immunoreactivity in cytoplasm or nucleus, positive determination application positive cell counting microscope * 400 magnification of each slice randomly selected 10 HPF, calculate the number of positive cells and the total cell number, calculate the percentage of protein imprinting. The gray bands were determined. The percentage of positive cells, Western blot gray value, the results of blood gas analysis and lung coefficient data are mean standard deviation ((?) + s) said that the group in different time were compared by t test.
experimental result
1, blood gas analysis (PO2)
There was no significant difference between the control group and the control group, and the LPS group was lower than the control group, with 1H and 2H as obvious. The improvement of PO2 in the rats with LPS induced lung injury was more obvious.
2, lung coefficient
The lung coefficient of rats in the injury group was higher than that in the control group. The pulmonary coefficient of the prevention and treatment group was lower than that of the injury group, and there was a significant difference.
3, gross specimen and HE staining results
The lung tissue of LPS group showed patchy necrosis, the pulmonary congestion, pulmonary interstitial edema, alveolar septal thickening, alveolar diffuse hemorrhage and scattered bronchial bleeding in the prophylaxis and treatment group had significantly reduced the lung surface hyperemia, patchy necrosis disappeared, with preventive medication group group.LPS was found in the alveolar a large number of red blood cells and neutrophil infiltration around blood vessels and bronchi loose tissue edema, more inflammatory cell infiltration by neutrophils, late scattered formed on the transparent film. The pathological preventive medication group and Treatment Medical Group lung had significant improvements, but still showed obvious inflammatory cell infiltration.
4, HSP70 immunohistochemical staining results
The normal group HSP70 mainly distributed in the bronchial and bronchiolar epithelial cells, alveolar cells and macrophages scattered in increased expression of.LPS positive staining group, scattered in macrophages and infiltrating neutrophils positive staining, the positive number of alveolar epithelial cells slightly increased, the percentage of positive cells compared with the control group, there was significant difference between the group and the preventive medication. Treatment group the number of positive cells increased.
5, Western blot analysis (western-bloting method)
The HSP70 band was located near 70KD. The expression of HSP70 protein showed that the expression level of prophylactic medication group and treatment group was wider and wider than that of the control group and LPS group. There was a significant difference between the preventive medication group and the treatment group (P0.01).
discuss
Acute lung injury is the pathological features of neutrophil accumulation in lung tissue, intravascular coagulation, pulmonary capillary permeability increase induced pulmonary edema, which leads to the biological effects of hypoxia.LPS can also increase the variety of bioactive factors mediated by the exacerbation of lung injury and the clinical symptoms. Heat shock protein can improve cellular stress ability, resist effect of various kinds of damage factors, plays an important role in protecting cells, thereby reducing animal mortality. Preventive medication group increased HSP70 expression in the experimental study, immunohistochemistry positive cells and Western results were higher than those of acute lung injury group, and arterial oxygen pressure, lung coefficient and pathological lung injury the change is light, treatment group were improved to a certain extent, but in arterial oxygen pressure, lung coefficient and pathological changes of HSP70 and pathology of lung injury As the amount of a preventive medication group. In order to reveal the cells treated with HSP70 after pretreatment of rhubarb cells increased synthesis, but also that the rhubarb may by inducing HSP70 expression increased to protect lung tissue damage. The results of the study on the treatment of rhubarb injection in clinical application of time has guiding significance.
conclusion
The experimental results show that the LPS group of lung injury is obvious, in the prophylaxis group showed obvious improvement in lung function, treatment group is that the expression of HSP70 was higher than the LPS group, and HSP70 in bronchial epithelial, bronchial and vascular endothelial showed strong positive expression, that HSP70 might be involved in the reduction of vascular permeability and to ensure the normal ventilation pipe. The experiment results show that the injection of rhubarb before LPS can increase the expression of HSP70 in lung, lung protection has obvious function, but in the treatment group, the expression of HSP70 increased the amount of visible light, and the preventive medication group there were significant differences in at the same time the application of rhubarb injection to induce ALI induced HSP70 expression increased and can not significantly improve lung function. The results of the study on Rhubarb injection on clinical application The time of treatment is of guiding significance, and it also shows that the protective effect of rhubarb on ALI is likely to be achieved by inducing the increase in the expression of HSP70.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

【引证文献】