本文關(guān)鍵詞：內(nèi)毒素誘導(dǎo)急性肺損傷中熱休克蛋白的表達(dá)及大黃對HSP70表達(dá)的調(diào)控　出處：《中國醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 大黃 內(nèi)毒素 急性肺損傷 HSP70 免疫組織化學(xué) 蛋白印跡

【摘要】： 內(nèi)毒素誘導(dǎo)急性肺損傷中熱休克蛋白的表達(dá)及大黃對HSP70表達(dá)的調(diào)控 前言 急性呼吸窘迫綜合癥(ARDS)是臨床常見的危急重癥,其病理特征是中性粒細(xì)胞在肺內(nèi)聚集、血管內(nèi)凝血、肺毛細(xì)血管通透性增加致肺水腫,從而導(dǎo)致低氧血癥甚至死亡。 熱休克蛋白(heat shock protein.HSP)是一種應(yīng)激蛋白(sress protein.SP),同時它也是一種內(nèi)源性的保護(hù)物質(zhì),其中HSP70為最保守、最主要的一類,在大多數(shù)生物中含量最多,在細(xì)胞應(yīng)激后生成最為顯著。另外,研究表明大黃對LPS誘導(dǎo)的ALI具有保護(hù)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,從而起到保護(hù)肺臟,減輕肺損傷LPS誘導(dǎo)的ALI的作用。 大黃對肺臟的保護(hù)作用是否與HSP家族成員有關(guān),即大黃是否通過誘導(dǎo)肺內(nèi)HSP70的表達(dá)水平來保護(hù)肺組織,目前國內(nèi)外尚未見報道,而且HSP70表達(dá)水平在LPS誘導(dǎo)的ALI的動物模型中的時間規(guī)律性尚且不清。本實(shí)驗應(yīng)用免疫組織化學(xué)染色技術(shù)和蛋白印跡試驗等技術(shù)進(jìn)行研究大黃,并對大黃預(yù)防組和大黃治療組LPS誘導(dǎo)的肺損傷組織中HSP70的表達(dá)部位及表達(dá)水平進(jìn)行對比,從而證實(shí)大黃對LPS誘導(dǎo)的ALI的保護(hù)機(jī)制可能與HSP70有關(guān),并為臨床上應(yīng)用大黃治療急性肺損傷提供理論依據(jù)。 材料和方法 動物模型的建立及分組：健康wistar大鼠60只,隨機(jī)分成對照組、ALI組、大黃預(yù)防組、大黃治療組4組,每組15只。麻醉后,頸內(nèi)靜脈給0.9％生理鹽水或給,LPS,于不同時間,腹主動脈取血做血?dú)夥治?后脫頸椎處死動物,并取雙側(cè)肺臟標(biāo)本,進(jìn)行肺系數(shù)測定,而后進(jìn)行HE染色及免疫組織化學(xué)染色(SP法)以及Western-Blotting測定蛋白表達(dá)水平。 結(jié)果判定：免疫組織化學(xué)陽性物質(zhì)表達(dá)在胞漿或胞核中,陽性判定應(yīng)用陽性細(xì)胞計數(shù)：顯微鏡×400倍率下每張切片中隨機(jī)選擇10個高倍視野,計算其中陽性細(xì)胞數(shù)和總細(xì)胞數(shù),求出百分比。蛋白印跡結(jié)果進(jìn)行條帶灰度測定。陽性細(xì)胞百分比、蛋白印跡灰度值、血?dú)夥治鼋Y(jié)果和肺系數(shù)等數(shù)據(jù)資料均以均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示,組內(nèi)不同時間比較均采用t檢驗。 實(shí)驗結(jié)果 1、血?dú)夥治?PO2) 對照組各時間段無明顯差異,LPS組較對照組下降,以1h、2h為明顯。預(yù)防用藥組對LPS誘導(dǎo)的肺損傷大鼠的PO2改善較明顯。 2、肺系數(shù) 損傷組大鼠肺系數(shù)增高,與對照組存在顯著性差異,預(yù)防用藥組和治療用藥組肺系數(shù)低于損傷組,存在顯著性差異。 3、大體標(biāo)本和HE染色結(jié)果 LPS組肺組織可見斑片狀壞死,整個肺充血、肺間質(zhì)水腫,肺泡間隔增寬,肺泡腔內(nèi)彌漫性出血及散在支氣管內(nèi)出血,預(yù)防用藥及治療用藥組肺表面充血有明顯減輕,斑片狀壞死消失,以預(yù)防用藥組為明顯。LPS組肺泡腔內(nèi)可見大量紅細(xì)胞和中性粒細(xì)胞浸潤,血管及支氣管周圍組織疏松水腫,有較多炎細(xì)胞浸潤，以中性粒細(xì)胞為主,晚期可見散在透明膜形成。預(yù)防用藥組和治療用藥組肺臟的組織病理學(xué)有明顯改善,但仍可見較明顯的炎細(xì)胞浸潤。 4、HSP70免疫組織化學(xué)染色結(jié)果 正常組HSP70主要分布于支氣管及細(xì)支氣管上皮細(xì)胞,肺泡細(xì)胞和巨噬細(xì)胞可見散在陽性著色。LPS組表達(dá)增多,可見散在巨噬細(xì)胞和浸潤的中性粒細(xì)胞陽性著色,肺泡上皮細(xì)胞陽性數(shù)輕度增加,陽性細(xì)胞率與對照組相比存在顯著性差異。預(yù)防用藥組與治療用藥組陽性細(xì)胞數(shù)增加。 5、蛋白質(zhì)印跡分析(western-bloting方法) HSP70條帶位于70KD附近,HSP70蛋白量顯示,預(yù)防用藥組和治療用藥組表達(dá)量較對照組和LPS組加深、加寬。并且預(yù)防用藥組與治療用藥組之間存在顯著性差異(P0.01) 討論 急性肺損傷病理特征是中性粒細(xì)胞在肺內(nèi)聚集,血管內(nèi)凝血,肺毛細(xì)血管通透性增加致肺水腫,從而導(dǎo)致低氧血癥。LPS還可以增加由其介導(dǎo)的各種生物活性因子的生物效應(yīng),加重肺臟損傷和臨床癥狀。熱休克蛋白可以提高細(xì)胞的應(yīng)激能力,抵御各種損害因素的影響,起到保護(hù)細(xì)胞的作用,從而降低動物的死亡率。本實(shí)驗研究中預(yù)防用藥組HSP70表達(dá)增加,免疫組織化學(xué)陽性細(xì)胞率和Western結(jié)果均高于急性肺損傷組,而且大鼠動脈血氧分壓增高、肺系數(shù)降低、肺組織損傷的病理學(xué)改變較輕,治療用藥組雖有一定程度的改善,但在動脈血氧分壓增高、肺系數(shù)降低、肺組織損傷的病理學(xué)改變和HSP70表達(dá)量上較預(yù)防用藥組差。揭示細(xì)胞經(jīng)大黃預(yù)處理后細(xì)胞中HSP70合成增加,同時也表明了大黃可能通過誘導(dǎo)HSP70的表達(dá)增加來保護(hù)損傷的肺組織。本研究的實(shí)驗結(jié)果對臨床上應(yīng)用大黃注射液的治療的時間上具有指導(dǎo)性意義。 結(jié)論 本實(shí)驗研究表明,LPS組肺臟損傷較明顯,在預(yù)防用藥組可見明顯的肺臟功能的改善,治療用藥組也可見HSP70表達(dá)較LPS組增高,并且HSP70在支氣管、細(xì)支氣管粘膜上皮和血管內(nèi)皮均有強(qiáng)陽性表達(dá),說明了HSP70很可能參與了降低血管通透性和保證氣管的正常通氣。本實(shí)驗的研究結(jié)果表明在注射LPS之前預(yù)先給予大黃注射液可以增加肺組織中HSP70的表達(dá)量,具有明顯的肺臟保護(hù)功能,但在治療用藥組中,可見HSP70表達(dá)量的輕度增加,與預(yù)防用藥組存在顯著性差異,說明在誘導(dǎo)ALI同時應(yīng)用大黃注射液來誘導(dǎo)HSP70表達(dá)量的增多并不能明顯改善肺臟功能。本研究的實(shí)驗結(jié)果對臨床上應(yīng)用大黃注射液的治療的時間上具有指導(dǎo)性意義,也說明了大黃對ALI的保護(hù)作用很可能是通過誘導(dǎo)HSP70的表達(dá)增多而實(shí)現(xiàn)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.

【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R363

【引證文獻(xiàn)】

相關(guān)會議論文 前1條

1 劉晶晶;張貴君;高曉美;;大黃藥材藥理作用研究概況[A];中國商品學(xué)會第十五屆學(xué)術(shù)論壇論文集[C];2013年

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本文編號：1423329