CN108042239A - 一种无创肺纤维化模型的建立方法 - Google Patents
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Abstract
本发明涉及一种肺纤维化动物模型的建立方法,即通过鼻腔滴入PM2.5混悬液制备肺纤维化动物模型。其优点表现在:本发明首次提出PM2.5鼻腔滴入建立小鼠肺纤维化模型,并通过实验验证了可行性,连续8周PM2.5鼻腔滴入可诱导小鼠肺纤维化。本发明的肺纤维化模型的建立方法没有使用博来霉素,并非通过气管滴入,操作简便,创伤性小。
Description
技术领域
本发明涉及动物模型技术领域,具体地说,是一种无创肺纤维化模型的建立方法。
背景技术
随着工业化、城市化的进程,空气污染日益加剧,细颗粒物(PM2.5)作为最主要的空气污染物,严重威胁着人类健康。流行病学调查表明,空气中PM2.5浓度的升高与冠心病、慢性阻塞性肺疾病(COPD)等心肺***疾病的发病及症状加重密切相关。PM2.5的空气动力学粒径小,随呼吸进入肺部后,极易沉积于远端小气道和肺泡,并从气道上皮进入肺组织间隙、甚至进入血液循环,可诱导炎症反应和氧化应激损伤,引起血管通透性增高和肺组织损伤。调查表明长期暴露于PM2.5环境中(即使污染程度相对较轻),机体的肺功能也会不断下降,主要表现在FEV1和FVC的降低。
目前常用的肺纤维化模型以博来霉素气管内滴注为主,但此法诱导的肺纤维化有自限性,且该方法是经气管滴入,操作难度较高,具有创伤性。因此,非常有必要建立一种相对简便且创伤性小的方法。
中国专利文献CN107115343A公开了一种通过胺碘酮和盐酸萘甲唑啉采用加样枪通过鼻腔给药建立大鼠肺细胞纤维化模型的方法。中国专利文献CN107469069A公开了一种向恒河猴皮下注射百草枯溶液、气管滴注博来霉素溶液建立肺纤维化动物模型的方法。中国专利文献CN106309479A公开了一种采用温石棉纤维非暴露法气管滴注SPF级C57/BL6小鼠建立肺纤维化动物模型的方法。中国专利文献CN105311618A公开了一种应用介导肺纤维化发生的重要启动因子作为诱导剂,采用血管紧张素Ⅱ皮下注射建立肺纤维化动物模型的方法。中国专利文献CN104605958A公开了一种采用内窥镜引导下气管内灌注的滴注菌液方式制作肺纤维化动物模型的方法。
而有关PM2.5的专利、科技论文等医学方面的文献,主要是关于如何防治PM2.5引发疾病的报道,关于本发明的利用PM2.5鼻腔滴入建立小鼠肺纤维化模型的方法目前还未见报道。
发明内容
本发明的第一个目的是,针对现有技术中的不足,提供PM2.5的一种新用途。
本发明的第二个目的是,提供一种无创肺纤维化模型的建立方法。
为实现上述第一个目的,本发明采取的技术方案是:PM2.5在制备肺纤维化动物模型中的应用。
进一步地,PM2.5混悬液在制备肺纤维化动物模型中的应用。
进一步地,所述的PM2.5混悬液为PM2.5用PBS缓冲液配制而成。
进一步地,所述的动物为啮齿类动物。
进一步地,所述的啮齿类动物为小鼠。
为实现上述第二个目的,本发明采取的技术方案是:一种肺纤维化动物模型的建立方法,通过鼻腔滴入PM2.5混悬液制备肺纤维化动物模型。
进一步地,所述的PM2.5混悬液为PM2.5用PBS缓冲液配制而成。
进一步地,所述的PM2.5混悬液的制备方法为:使用PM2.5采样器,采用玻璃纤维滤膜,采集大气PM2.5,将采集到颗粒物的滤膜置于去离子水中,使用超声波清洗器洗脱下来,然后冷冻真空干燥,收集PM2.5,用PBS缓冲液配制成PM2.5混悬液,超声震荡混匀。
进一步地,所述的PM2.5混悬液剂量为7.8mg/kg,滴注体积为50μl,每周2次,连续滴注8周。
进一步地,所述的动物为小鼠。
本发明优点在于:
1、本发明首次提出PM2.5鼻腔滴入建立小鼠肺纤维化模型,并通过实验验证了可行性,连续8周PM2.5鼻腔滴入可诱导小鼠肺纤维化。PM2.5鼻腔滴入导致小鼠的吸气量降低、肺顺应性降低和呼吸气流阻塞,BALF中炎症细胞总数增加,肺部组织中炎症积分增加、气道胶原沉积厚度增加、肺组织羟脯胺酸含量增加,同时平均肺泡内衬间隔无改变。
2、本发明的肺纤维化模型的建立方法没有使用博来霉素,并非通过气管滴入,操作简便,创伤性小。
附图说明
附图1是对照组肺组织的HE染色图片。
附图2是PM2.5滴入组肺组织的HE染色图片。
附图3是对照组气管的Masson染色图片。
附图4是PM2.5滴入组气管的Masson染色图片。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
实施例1
材料与方法
一、材料
8-10周SPF级C57/BL6小鼠,体重22-25g,购自上海西必普凯公司,饲养于本院动物中心的屏障***,本研究得到医院伦理委员会的批准。小动物气体麻醉机由上海玉研仪器公司提供。异氟烷由上海玉研仪器公司提供。肺功能仪(Forced Maneuvers***)由英国EMMS公司提供。细胞离心机(ShandonCytospin)由美国Thermo Fisher公司提供。刘氏染液由珠海贝索生物技术有限公司提供。羟脯氨酸试剂盒购于南京建成生物工程研究所。
二、大气PM2.5采样及处理
2016年10月到2017年4月,于上海市非工业区使用崂应2030型中流量PM2.5采样器(青岛崂山应用技术研究所)采用玻璃纤维滤膜(青岛崂山应用技术研究所)采集大气PM2.5,将采集到颗粒物的滤膜置于去离子水中,使用超声波清洗器洗脱下来,然后冷冻真空干燥,收集PM2.5,-20℃干燥保存。使用前,称取一定量的PM2.5,用PBS缓冲液配制成PM2.5混悬液,超声震荡混匀,4℃保存。
三、方法
1.PM2.5暴露:16只C57/BL6小鼠在动物实验室内饲养一周后,随机分为两组分笼饲养,分别标记为对照组、PM2.5鼻腔滴入组,每组8只。小鼠置于气体麻醉机密封箱中,给予异氟烷麻醉,模型组鼻腔滴入PM2.5混悬液,剂量为7.8mg/kg,滴注体积为每只50μl。对照组小鼠鼻腔内滴入生理盐水50μl。每周两次,连续滴注8周。
2.肺功能检测:腹腔注射0.15-0.2ml的1%戊巴比妥钠150-200μl后,小鼠气管切开,***气管导管,置入体积描记箱(Forced Maneuvers***),并连接电脑控制的呼吸机,呼吸频率150次/分。测量吸气量(IC)、功能残气量(FRC)、肺总量(TLC)、用力呼气量(FVC)、第20ms、50ms的用力呼气量(FEV25,FEV50)、顺应性(Cchord)。
3.支气管肺泡灌洗液(BALF):腹腔注射0.4ml的1%戊巴比妥钠以处死小鼠。应用2ml的PBS进行支气管肺泡灌洗,回收BALF。在心脏的右心室穿刺获得血液。BALF离心,细胞颗粒重悬,计数后,在细胞离心机制片,采用刘氏染液染色。显微镜下计数至少500个细胞,并区分各类炎症细胞的比例。
4.组织病理分析:左肺应用4%***灌注、固定,石蜡包埋,切片,片厚4μm,进行HE染色及Masson染色。HE染色评估支气管周围的肺部炎症积分。0=无炎症反应,1=轻度炎症反应,支气管壁、血管壁或肺泡间隔有少量炎症细胞,2=中度炎症反应,支气管壁、血管壁、肺泡间隔有成片炎症,3=重度炎症反应,支气管壁、血管壁、肺泡间隔有广泛的炎症。在显微镜下,测量平均内衬间隔(Lm),应用目镜测微尺,计数5条线(长度500μm)上的肺泡间隔数。Lm(μm)=500/肺泡间隔数。Masson染色后,通过Image J图像分析软件测量气道上皮下胶原沉积面积、气道基底膜周长,计算气道上皮下胶原沉积厚度(Wcol/Pbm)。
5.肺组织羟脯氨酸的测定:用碱水解法按试剂盒说明书操作测定肺组织羟脯氨酸含量,计算公式:羟脯氨酸含量(μg/mg湿重)=(测定管吸光度-空白管吸光度)/(标准管吸光度-空白管吸光度)×标准管含量((5μg/ml)×水解液总体积(10ml)/组织湿重(mg)。
四、统计学处理
数据以表示。采用SPSS 17.0统计软件进行统计分析,两组数据之间的差异性进行t检验。P<0.05表示差异有显著性。
结果
一、肺功能
PM2.5鼻腔滴入组小鼠的IC、Cchord、FEV25/FVC、FEV50/FVC低于对照组小鼠,FRC高于对照组小鼠,肺总量无变化。结果见表1。
表1各组肺功能参数
对照组 | PM2.5滴入组 | |
IC | 0.51±0.08 | 0.31±0.03*** |
FRC | 0.36±0.03 | 0.56±0.05*** |
Cchord | 0.018±0.02 | 0.012±0.04* |
FEV25/FVC(%) | 62.35±6.79 | 41.37±6.32*** |
FEV50/FVC(%) | 91.62±3.29 | 80.49±4.59** |
注:与8周对照组小鼠相比,*P<0.05,**P<0.01,***P<0.001。
二、BALF细胞计数
PM2.5滴入组小鼠BAL液中的细胞总数、巨噬细胞数、中性粒细胞数、嗜酸性粒细胞数及淋巴细胞数均高于对照组小鼠。结果见表2。
表2各组BALF细胞计数(×103/ml)
对照组 | PM2.5滴入组 | |
细胞总数 | 224±31.9 | 649±74.53** |
巨噬细胞 | 201.3±34.4 | 476.12±90.03** |
淋巴细胞 | 16.53±4.35 | 90.33±45.14** |
中性粒细胞 | 2.20±2.23 | 56.37±29.42** |
嗜酸性粒细胞 | 3.96±1.52 | 26.18±8.23** |
注:与8周对照组小鼠相比,*P<0.05,**P<0.01。
三、肺部炎症积分和Lm
肺组织HE染色结果表明,PM2.5鼻腔滴入组小鼠肺部气道周围炎症细胞浸润明显,肺部炎症积分高于对照组小鼠。PM2.5鼻腔滴入组小鼠Lm与对照组小鼠无差别。典型的肺组织图片见附图1(对照组)、附图2(PM2.5滴入组),结果见表3。
四、气道上皮下胶原沉积厚度
肺组织Masson染色结果表明,PM2.5鼻腔滴入组小鼠气道周围胶原沉积相比对照组明显增多,即气道上皮下胶原沉积厚度(Wcol/Pbm)较对照组小鼠显著增高。典型的Masson染色图片见附图3(对照组)、附图4(PM2.5滴入组),结果见表3。
五、肺组织羟脯氨酸含量
与对照组小鼠相比,PM2.5鼻腔滴入组小鼠肺组织内羟脯氨酸含量明显增高。结果见表3。
表3各组肺部炎症积分、胶原沉积厚度及肺组织羟脯氨酸含量
对照组 | PM2.5滴入组 | |
炎症积分 | 0.26±0.03 | 1.28±0.35** |
Lm | 85.01±15.06 | 81.45±17.23 |
胶原(μm2/μm) | 2.16±1.40 | 9.14±3.12** |
羟脯氨酸(μg/g) | 323.83±232.71 | 805.87±126.22** |
注:与8周对照组小鼠相比,*P<0.05,**P<0.01。
在本研究中,8周的间断PM2.5鼻腔滴入引起小鼠肺组织出现明显的炎症反应,表现为BALF中总的细胞计数、巨噬细胞、中性粒细胞、淋巴细胞及嗜酸性粒细胞计数增加,肺部炎症积分增加,肺组织切片可观察到支气管、血管周围炎性细胞浸润。巨噬细胞可分泌促炎因子和趋化因子,如GM-CSF、CCL2、CXCL8、TGF-β等,可驱动肺组织嗜中性粒细胞募集和巨噬细胞的成熟,还能上调CD204水平而促进CCL18、CCL2和IL-1ra的表达,导致胶原纤维的合成增多。有临床分析指出,肺纤维化患者的肺泡灌洗液中淋巴细胞数往往明显增多,这可能和T淋巴细胞介导的免疫失衡导致慢性炎症持续发展,组织修复不协调,成纤维细胞增生,引起细胞外基质分泌增多相关。
肺纤维化的病理特征表现为长期的肺部炎症导致肺泡持续损伤,成纤维细胞的增生、转型和大量细胞外基质堆积,从而导致肺组织的反复破坏、修复,小气道壁和肺泡壁及肺小血管壁大量胶原沉积,最终形成肺纤维化。通过Masson染色测量气道上皮下胶原沉积厚度(Wcol/Pbm),结果表明PM2.5滴入组小鼠的气道上皮下胶原沉积量高于对照组。同时发现,PM2.5滴入组小鼠肺组织羟脯含量明显高于对照组。而羟脯胺酸是胶原纤维的重要组成成分之一,其含量的增高也提示肺组织胶原纤维的含量增高。为了评估PM2.5鼻腔滴入是否介导形成肺气肿,本研究测量肺泡平均内衬间隔(Lm),结果发现,PM2.5鼻腔滴入组小鼠的肺泡Lm和对照组相比并无明显差别,表明PM2.5鼻腔滴入并未诱导肺气肿。
PM2.5暴露与人体的肺功能下降密切相关。随着环境PM2.5浓度的升高,人的肺功能指标如FVC、FEV1/FVC及FEF25-75也会随之下降。而肺纤维化时,肺功能往往相应降低,表现为限制性通气功能障碍。通过检测小鼠肺功能发现,与对照组小鼠相比,PM2.5滴入组小鼠的IC降低,肺弹性回缩力增高和肺顺应性降低,表现为Cchord降低,呼出气流阻塞,表现为FEV25/FVC,FEV50/FVC下降。因此,本研究证实,8周的间断PM2.5鼻腔滴入介导限制性通气功能障碍和气流阻塞。
综上所述,8周间断PM2.5鼻腔滴入成功诱导小鼠出现肺纤维化模型。
实施例2
PM2.5混悬液制备方法同实施例1,向大鼠鼻腔滴入PM2.5混悬液,制备大鼠肺纤维化模型。
实施例3
PM2.5混悬液制备方法同实施例1,向豚鼠鼻腔滴入PM2.5混悬液,制备豚鼠肺纤维化模型。
实施例4
PM2.5混悬液制备方法同实施例1,向家兔鼻腔滴入PM2.5混悬液,制备家兔肺纤维化模型。
实施例5
PM2.5混悬液制备方法同实施例1,向猴子鼻腔滴入PM2.5混悬液,制备猴子肺纤维化模型。
实施例6
PM2.5混悬液制备方法同实施例1,向狗鼻腔滴入PM2.5混悬液,制备狗肺纤维化模型。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (10)
1.PM2.5在制备肺纤维化动物模型中的应用。
2.PM2.5混悬液在制备肺纤维化动物模型中的应用。
3.根据权利要求2所述的应用,其特征在于,所述的PM2.5混悬液为PM2.5用PBS缓冲液配制而成。
4.根据权利要求2所述的应用,其特征在于,所述的动物为啮齿类动物。
5.根据权利要求4所述的应用,其特征在于,所述的啮齿类动物为小鼠。
6.一种肺纤维化动物模型的建立方法,其特征在于,通过鼻腔滴入PM2.5混悬液制备肺纤维化动物模型。
7.根据权利要求6所述的建立方法,其特征在于,所述的PM2.5混悬液为PM2.5用PBS缓冲液配制而成。
8.根据权利要求6所述的建立方法,其特征在于,所述的PM2.5混悬液的制备方法为:使用PM2.5采样器,采用玻璃纤维滤膜,采集大气PM2.5,将采集到颗粒物的滤膜置于去离子水中,使用超声波清洗器洗脱下来,然后冷冻真空干燥,收集PM2.5,用PBS缓冲液配制成PM2.5混悬液,超声震荡混匀。
9.根据权利要求6所述的建立方法,其特征在于,所述的PM2.5混悬液剂量为7.8mg/kg,滴注体积为50μl,每周2次,连续滴注8周。
10.根据权利要求6所述的建立方法,其特征在于,所述的动物为小鼠。
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN112868596A (zh) * | 2020-10-30 | 2021-06-01 | 广州医科大学附属第一医院(广州呼吸中心) | 肺纤维化大鼠模型、其建立方法及评估方法 |
CN112568188A (zh) * | 2021-02-24 | 2021-03-30 | 澎立生物医药技术(上海)有限公司 | 一种制作Brown Norway大鼠肺纤维化模型的方法 |
CN115414346A (zh) * | 2022-08-29 | 2022-12-02 | 湖南复瑞生物医药技术有限责任公司 | 一种胺碘酮的新应用方法 |
CN115414346B (zh) * | 2022-08-29 | 2023-09-29 | 湖南复瑞生物医药技术有限责任公司 | 一种应用胺碘酮构造肺纤维化模型的方法 |
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