不同模型复制方式致肺结节小鼠模型的比较研究

A comparative study on different modeling methods for inducing pulmonary sarcoidosis in mice

摘要:

目的 比较多壁碳纳米管（MWCNT）、MWCNT+脂多糖（LPS）气管滴注、MWCNT+LPS雾化3种方法复制的肺结节小鼠模型，为后续研究提供依据。方法 将40只雄性C57BL/6小鼠随机分为空白组、MWCNT组、MWCNT+LPS气管滴注组与MWCNT+LPS雾化组，每组10只。空白组小鼠气管滴注同体积生理盐水，其余各组均气管滴注60 μL 8 mg/kg MWCNT悬液，在此基础上，MWCNT+LPS气管滴注组联合进行气管滴注LPS溶液（5 mg/kg， 7 d/次），MWCNT+LPS雾化组联合进行雾化吸入LPS溶液（5 mg/mL，30 min/次，7 d/次），共28 d，第29天取材。检测小鼠肺功能［潮气量（TV）、每分钟通气量（MV）、特殊气道阻力（sRaw）、功能残气量（FRC）］，观察小鼠肺部影像学及组织病理学，免疫组织化学法检测巨噬细胞标志物F4/80和淋巴细胞标志物CD3⁺T蛋白表达，检测支气管肺泡灌洗液（BALF）中炎症细胞分类计数，LDH和BCA法检测肺损伤，血细胞分类法检测白细胞比例，酶联免疫吸附试验检测肺泡灌洗液（BALF）中干扰素-γ（IFN-γ）、肿瘤坏死因子-α（TNF-α）与白细胞介素-2（IL-2）水平。结果 各组小鼠的TV、MV及FRC水平比较，差异均无统计学意义（P >0.05）；各组小鼠sRaw水平比较，差异有统计学意义（P <0.05），MWCNT+LPS气管滴注组小鼠sRaw水平增加。肺部影像学显示，MWCNT组小鼠右肺可见一类圆形实性结节，MWCNT+LPS气管滴注组小鼠左肺可见片状磨玻璃样改变，MWCNT+LPS雾化组右肺可见一单发小斑片状改变。肺部组织病理学显示，空白组小鼠肺部光滑质嫩；MWCNT组小鼠肺部有黑色沉积影，表面有白色小结节；MWCNT+LPS气管滴注组肉眼可见大量黑色碳纳米粒沉积物，伴有大量白色结节形成；MWCNT+LPS雾化组小鼠肺部质嫩，中央部有大量黑色沉积物，可见团状小结节。HE染色显示，空白组小鼠肺泡管和肺泡规整，MWCNT组小鼠肺脏可见多灶性轻微肉芽肿，MWCNT+LPS气管滴注组小鼠肺脏可见中度多灶性肉芽肿，MWCNT+LPS雾化组小鼠肺脏可见多灶性轻微肉芽肿。免疫组织化学染色结果显示，与空白组相比，MWCNT组小鼠F4/80和CD3⁺T均在肺组织中少量浸润；MWCNT+LPS气管滴注组的F4/80呈灶状浸润，胞质染色强阳性，CD3⁺T细胞呈局灶性分布；MWCNT+LPS雾化组的F4/80与CD3⁺呈弥散性浸润，在组织间质中广泛分布。与空白组相比，MWCNT组、MWCNT+LPS气管滴注组、MWCNT+LPS雾化组小鼠BALF中性粒细胞数均增加（P <0.05），MWCNT+LPS气管滴注组出现大量巨噬细胞。各组小鼠BALF的LDH值比较，差异有统计学意义（P <0.05）；与空白组相比，MWCNT组LDH值差异无统计学意义（P >0.05），MWCNT+LPS气管滴注组LDH值升高（P <0.05）。各组小鼠BALF的BCA比较，差异无统计学意义（P >0.05）。与空白组相比，MWCNT+LPS气管滴注组的单核细胞和嗜酸性粒细胞计数升高（P <0.05）；MWCNT+LPS雾化组小鼠嗜酸性粒细胞计数升高（P <0.05），中性粒细胞、淋巴细胞及单核细胞比较，差异无统计学意义（P >0.05）。与空白组相比，MWCNT组TNF-α水平升高（P <0.05），INF-γ、IL-2水平比较，差异无统计学意义（P >0.05）；MWCNT+LPS气管滴注组的INF-γ、TNF-α、IL-2水平均升高（P <0.05）；MWCNT+LPS雾化组的INF-γ、TNF-α、IL-2水平也均升高（P <0.05）。结论 MWCNT、MWCNT+LPS滴注和MWCNT+LPS雾化3种方法均可成功复制肺结节模型，其中MWCNT+LPS雾化方法复制的肺结节模型能够有效模拟人类结节病理生理学的多个关键特征，是肺结节机制研究和药物开发的有价值实验模型。

Abstract:

Objective By comparing three construction methods of mouse lung sarcoidosis models, which concludes MWCNTs, MWCNTs + lipopolysaccharide (LPS) intratracheal instillation, and MWCNTs + LPS aerosolization to provide a basis for subsequent research.Methods Forty male C57BL/6 mice were randomly divided into a control group, an MWCNT group, an MWCNT + LPS tracheal instillation group, and an MWCNT + LPS nebulization group, with 10 mice in each group. Mice in the control group received tracheal instillation of the same volume of physiological saline, while mice in the other groups received tracheal instillation of 60 μL of an 8 mg/kg MWCNT suspension. In addition, the MWCNT + LPS tracheal instillation group received concurrent tracheal instillation of LPS solution (5 mg/kg, 7 days per dose). and the MWCNT + LPS nebulization group received nebulization of LPS solution (5 mg/mL, 30 minutes per session, 7 days per week). The treatment lasted for 28 days, and tissue samples were collected on day 29. Mouse lung function was assessed [tidal volume (TV), minute ventilation (MV), specific airway resistance (sRaw), functional residual capacity (FRC) ], and lung imaging and histopathology were observed. Immunohistochemistry was used to detect the expression of macrophage marker F4/80 and lymphocyte marker CD3⁺T protein. Inflammatory cell counts in bronchoalveolar lavage fluid (BALF) were determined, and lung injury was assessed using LDH and BCA assays. White blood cell proportions were analyzed by blood cell classification, and levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-2 (IL-2) in BALF were measured by ELISA.Results Comparisons of TV, MV, and FRC levels among the groups showed no statistically significant differences (P > 0.05); however, comparisons of sRaw levels among the groups revealed statistically significant differences (P < 0.05), with increased sRaw levels observed in the MWCNT + LPS tracheal instillation group. Pulmonary imaging revealed that the right lung of the MWCNT group showed a single round solid nodule, the left lung of the MWCNT + LPS tracheal instillation group showed patchy ground-glass opacities, and the right lung of the MWCNT + LPS nebulization group showed a single small patchy lesion. Pulmonary histopathology showed that the lungs of the control group were smooth and tender; MWCNT group mice had black deposits in the lungs with white sarcoidosis on the surface; MWCNT + LPS tracheal instillation group mice had a large amount of black carbon nanotube deposits visible to the naked eye, accompanied by the formation of numerous white sarcoidosis; MWCNT + LPS nebulization group mice had tender lungs with a large amount of black deposits in the central region and visible nodular lesions. HE staining showed that the alveolar tubes and alveoli of the control group mice were well-organized; the lungs of the MWCNT group mice exhibited multifocal mild granulomas; the lungs of the MWCNT + LPS tracheal instillation group mice exhibited moderate multifocal granulomas; and the lungs of the MWCNT + LPS nebulization group mice exhibited multifocal mild granulomas. Immunohistochemical staining results showed that compared with the control group, F4/80 and CD3⁺T cells were slightly infiltrated in the lung tissue of the MWCNT group; in the MWCNT + LPS tracheal instillation group, F4/80 showed focal infiltration with strong cytoplasmic staining, and CD3⁺T cells were distributed in focal areas; In the MWCNT + LPS aerosol group, F4/80 and CD3⁺ cells showed diffuse infiltration and were widely distributed in the interstitial tissue. BALF cell classification showed that, compared with the blank group, the number of neutrophils in the BALF of mice in the MWCNT group, MWCNT + LPS tracheal instillation group, and MWCNT + LPS nebulization group significantly increased (P < 0.05), and a large number of macrophages appeared in the MWCNT + LPS tracheal instillation group. Lung injury results showed that there were statistically significant differences in LDH levels in BALF among the groups (P < 0.05). Compared with the control group, there was no statistically significant difference in LDH levels in the MWCNT group (P > 0.05), while LDH levels were elevated in the MWCNT + LPS tracheal instillation group (P < 0.05). There were no statistically significant differences in BCA levels in BALF among the groups (P > 0.05). Peripheral blood leukocyte differential counts showed that, compared with the control group, the MWCNT + LPS tracheal instillation group had elevated monocyte and eosinophil counts (P < 0.05); Eosinophil counts were elevated in the MWCNT + LPS nebulization group (P < 0.05), while there were no statistically significant differences in neutrophil, lymphocyte, and monocyte counts (P > 0.05). ELISA results showed that, compared with the control group, TNF-α levels were elevated in the MWCNT group (P < 0.05), while there were no statistically significant differences in INF-γand IL-2 levels (P > 0.05); In the MWCNT + LPS tracheal instillation group, levels of INF-γ, TNF-α, and IL-2 were all elevated (P < 0.05); in the MWCNT + LPS aerosol group, levels of INF-γ, TNF-α, and IL-2 were also all elevated (P < 0.05).Conclusions All three methods, MWCNT, MWCNT + LPS tracheal instillation and MWCNT + LPS aerosolization, could successfully construct lung sarcoidosis models. Among which the mouse lung sarcoidosis model constructed by the MWCNT + LPS nebulization method was able to effectively mimic a number of key features of the pathophysiology of human sarcoidosiss, which provided a valuable experimental model for the study of the mechanism of lung sarcoidosiss and drug development.