大鼠结核性胸膜炎模型和胸腔炎症免疫反应的研究

A study on the model of tuberculous pleurisy and intrapleural inflammatory and immunological responses in rats

目的探讨建立大鼠结核性胸膜炎模型的方法以及观察胸腔局部炎症免疫反应.方法用标准人型结核分枝杆菌菌株H37Rv 0.03 mg注入50只Wistar大鼠胸腔内,并在注入后第1、2、3、5、7、10、15、20、30、60天分批处死实验动物.解剖胸腔,记录胸腔积液量,观察胸腔、胸膜和肺组织大体及镜下病理变化.检测胸腔积液中白细胞数及分类、总蛋白质(TP)、葡萄糖(GLU)和乳酸脱氢酶(LDH)的含量,测定可溶性细胞间黏附分子1(sICAM-1)、转移生长因子β1(TGF-β1)和γ干扰素(IFN-γ)的水平.另对10只大鼠胸腔内注入2 ml生理盐水,10只注入2 ml蛋白纯化衍生物(PPD)原液作为对照.结果大鼠胸腔内注入结核分枝杆菌后15 d内均有双侧胸腔积液,胸腔积液量于第5天最多(6.7±0.5)ml.胸腔积液白细胞数第1天最高(10.3×109/L),随时间而下降,第15天为3.4×109/L.细胞分类第1天中性粒细胞占优势(66%),以后淋巴细胞明显增多,第15天淋巴细胞占92%.TP为51～55 g/L.葡萄糖从第1天的5.2 mmol/L逐渐降低至第15天的2.8 mmol/L.LDH在第1天为18.1 μmol·s-1·L-1,此后随时间持续升高,第15天为28.9 μmol·s-1·L-1.胸腔积液的生化指标符合结核性胸腔积液的特征.sICAM-1水平早期升高,第7天后下降并低于第1天的水平.IFN-γ水平第1天为41.2 pg/ml,以后持续升高并维持在较高水平.TGF-β1第7天最高(47.2 ng/ml),第15天时已下降至低于第1天的水平.而IFN-γ/TGF-β1比值持续上升,从第1天的1.32升至第15天的5.69.相关分析显示sICAM-1和IFN-γ与白细胞数、分类和LDH水平关系较为密切.病理组织改变显示早期胸膜炎症至后期干酪性坏死的病理过程.结论 Wistar大鼠可作为结核性胸膜炎的实验动物.急性结核性胸膜炎的胸腔局部增强/抑制结核病炎症免疫反应以增强为主.

Objectie To deelop a rat model of tuberculous pleurisy and to explore the mechanism of intrapleural inflammatory and immunological responses. Methods Fifty Wistar rats were injected intrapleurally with 0.03 mg of standard human mycobacterium tuberculous bacilli H 37R each. The rats were killed in group on days 1, 2, 3, 5, 7, 10, 15, 20, 30 and 60 after the day of intrapleural injection. The thorax was opened and the amount of pleural effusion was recorded, and histopathology of pleural tissues and lung tissues were obsered. The white blood cell (WBC) count and differentials, leels of total protein(TP), glucose(GLU) and lactic dehydrogenase (LDH) of pleural effusions were determined. Pleural fluid was analyzed for the leels of soluble intercellular adhesion molecule-1 (sICAM-1), transforming growth factor β 1 (TGF-β 1) and interferon γ (IFN-γ) by using appropriate bioassays. Ten rats were intrapleurally receied 2 ml of normal saline and another 10 rats receied 2 ml of undiluted PPD solution each as control. Results Bilateral pleural effusions appeared within 15 days in all rats intrapleurally receied tuberculous bacilli. The peak amount of pleural fluid was on day 5 (6.7±0.5 ml). The neutrophils were the predominant cells for the first 24 hours, and then were followed by lymphocytes. In the pleural fluid, total protein concentration was between 51-55 g/L. The leels of glucose and LDH were 5.2 mmol/L and 18.1 μmol·s -1·L -1 on day 1 and changed to 2.8 mmol/L and 28.9 μmol·s -1·L -1 on day 15 respectiely. The biochemistry parameters were in accordance with characteristics of tuberculous pleurisy. The sICAM-1 leel increased early (21.9 ng/ml on day 1) and peaked on day 3 (38.0 ng/ml), then decreased oer time (4.4 ng/ml on day 15). The leel of IFN-γ was 41.2 pg/ml on day 1 and increased and maintained at high leels oer time. TGF-β 1 leels increased and peaked on day 7 (47.2 ng/ml), and then on day 15 decreased to a leel lower than that of day 1. The ratio of IFN-γ/TGF-β 1 increased from 1.32 on day 1 to 5.69 on day 15. Correlation analysis showed that sICAM-1 and IFN-γ were closely related with WBC count and its differentials, as well as with LDH leels. Histopathological study reealed early pleural inflammation and late caseation. Conclusions Wistar rats can be used as an experimental model for tuberculous pleurisy. Tuberculous inflammatory and immunological responses in acute tuberculous pleurisy is enhanced rather than suppressed.